Plants With Useful Traits and Related Methods

ABSTRACT

The present invention provides methods for obtaining plants that exhibit useful traits by transient suppression of the MSH1 gene of the plants. Methods for identifying genetic loci that provide for useful traits in plants and plants produced with those loci are also provided. In addition, plants that exhibit the useful traits, parts of the plants including seeds, and products of the plants are provided as well as methods of using the plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/540,236, filed Sep. 28, 2011 andincorporated herein by reference in its entirety, and the benefit under35 U.S.C. §119(e) of U.S. Provisional Application No. 61/481,519, filedMay 2, 2011 and incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under a grant from theDepartment of Energy (DE-FG02-07ER15564 and DE-FG02-10ER16189) and theNational Science Foundation (IOS 0820668 and IOS 1126935). Thegovernment has certain rights to this invention.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named“46589_(—)103288_SEQ_LST_ST25.txt”, which is 75,938 bytes in size(measured in operating system MS-Windows) and was created on May 1,2012, is contemporaneously filed with this specification by electronicsubmission (using the United States Patent Office EFS-Web filing system)and is incorporated herein by reference in its entirety.

BACKGROUND OF INVENTION

The MSH1 gene represents a MutS homolog that has undergone at least twoimportant changes in gene structure within land plants (Abdelnoor et al.2003). MutS is a prokaryotic gene that participates in mismatch repairand suppression of homologous recombination. Consistent with a model ofdirect protein-DNA interaction, MSH1 encodes not only DNA binding(Domain I) and ATPase (Domain V) domains, but has undergone gene fusionearly in its evolution to acquire a carboxy-terminal GIY-YIG typeendonuclease domain (Domain VI) (Abdelnoor et al. 2006). The protein hasalso gained domains II, III, and IV, appearing well-conserved among allland plants. This complexity of gene structure suggests that MSH1 hasacquired new functions in plants. While numerous MutS homologs arecharacterized in eukaryotic lineages, no gene outside of land plants hasbeen found to display the unusual features of MSH1.

MSH1 function has been studied in Arabidopsis with MSH1 null (EMS andT-DNA insertion) mutants (i.e. msh1 mutants) and in other plant speciesby MSH1 RNAi suppression (Sandhu et al. 2007; Xu et al. 2011). Whatemerged from these studies is that the phenotypic consequences of RNAisuppression are quite similar among species, including leaf variegation,cytoplasmic male sterility (CMS), a reduced growth-rate phenotype,delayed or non-flowering phenotype, and enhanced susceptibility topathogens. Exposure to heat (Shedge et al. 2010), high light stress (Xuet al. 2011) and other environmental stress conditions (Hruz et al.2008) result in markedly reduced MSH1 transcript levels.

Initial MSH1 investigations suggested its direct influence on plantmitochondrial genome stability. Null msh1 mutants in Arabidopsis displayenhanced recombination activity at 47 mitochondrial repeats that, overmultiple generations, creates significant genomic rearrangement. Agenomic consequence of MSH1 disruption is the process ofsubstoichiometric shifting (SSS) (Arrieta-Montiel et al. 2009). SSSactivity produces dramatic changes in relative copy number of parts ofthe mitochondrial genome, causing selective amplification or suppressionof genes residing on affected subgenomes. There are phenotypicconsequences to these genomic changes; the SSS process participates inexpression of cytoplasmic male sterility (Sandhu et al. 2007), as wellas its spontaneous reversion to fertility in natural populations (Janskaet al. 1998; Bellaoui et al. 1998; Davila et al. 2011; Mackenzie, 2011).In fact, MSH1 may have played a role in the evolution of gynodioecy as areproductive strategy in plants (McCauley and Olson, 2008).

Prior to its cloning and identification as a MutS homolog, the MSH1 genewas first named Chloroplast Mutator (CHM) by G. Redei, because itsmutation resulted in variegation and altered growth that appeared toderive from chloroplast dysfunction (Redei 1973). In fact, MSH1 encodesa dual targeted protein. A MSH1-GFP transgene fusion protein localizesto both mitochondrial and plastid nucleoids (Xu et al. 2011). Thenucleoid is a small, dense protein-RNA-DNA complex that envelopes theorganellar genomes. Unlike the mitochondrion however, whererecombination is prevalent, no evidence of enhanced chloroplastrepeat-mediated recombination is observed in the msh1 mutant. It ispossible that MSH1 disruption affects replication features of theplastid genome.

In summary, the effects of MSH1 suppression that have been disclosed inthe aforementioned references are limited to effects on plantmitochondria and plastids.

Evidence exists in support of a link between environmental sensing andepigenetic changes in both plants and animals (Bonasio et al., Science330, 612, 2010). Trans-generational heritability of these changesremains a subject of active investigation (Youngson et al. Annu. Rev.Genom. Human Genet. 9, 233, 2008). Previous studies have shown thataltered methylation patterns are highly heritable over multiplegenerations and can be incorporated into a quantitative analysis ofvariation (Vaughn et al. 2007; Zhang et al. 2008; Johannes et al. 2009).Earlier studies of methylation changes in Arabidopsis suggestamenability of the epigenome to recurrent selection and also suggestthat it is feasible to establish new and stable epigenetic states (F.Johannes et al. PLoS Genet. 5, e1000530 (2009); F. Roux et al. Genetics188, 1015 (2011). Manipulation of the Arabidopsis met1 and ddmt mutantshas allowed the creation of epi-RIL populations that show bothheritability of novel methylation patterning and epiallelic segregation,underscoring the likely influence of epigenomic variation in plantadaptation (F. Roux et al. Genetics 188, 1015 (2011)). In naturalpopulations, a large proportion of the epiallelic variation detected inArabidopsis is found as CpG methylation within gene-rich regions of thegenome (C. Becker et al. Nature 480, 245 (2011), R. J. Schmitz et al.Science 334, 369 (2011).

SUMMARY OF INVENTION

Methods for producing a plant exhibiting useful traits, methods foridentifying one or more altered chromosomal loci in a plant that canconfer a useful trait, methods for obtaining plants comprising modifiedchromosomal loci that can confer a useful trait, plants exhibiting theuseful traits, parts of those plants including cells, leafs, stems,flowers and seeds, methods of using the plants and plant parts, andproducts of those plants and plant parts, including processed productssuch as a feed or a meal are provided herein.

In certain embodiments, methods for producing a plant exhibiting auseful trait comprising the steps of: a). suppressing expression of MSH1gene(s) in a first parental plant or plant cell; b). outcrossing theparental plant of step (a), progeny of the parental plant of step (a), aplant obtained from the plant cell of step (a), or progeny of a plantobtained from the plant cell of step (a) to a second plant wherein MSH1had not been suppressed; c). screening a population of progeny plantsobtained from the outcross of step (b) for at least one useful trait,wherein a portion of the population of progeny plants express MSH1; and,d). selecting a progeny plant comprising the trait that expresses MSH1,wherein the trait is heritable and reversible, are provided. In certainembodiments of the methods, the trait is associated with one or morealtered chromosomal loci. In certain embodiments, such alteredchromosomal loci can comprise loci that are methylated. In certainembodiments, methods for producing a plant exhibiting a useful traitcomprising the steps of: a). suppressing expression of MSH1 gene(s) in afirst parental plant or plant cell; b). outcrossing the parental plantof step (a), progeny of the parental plant of step (a), a plant obtainedfrom the plant cell of step (a), or progeny of a plant obtained from theplant cell of step (a) to a second plant wherein MSH1 had not beensuppressed; c). screening a population of progeny plants obtained fromthe outcross of step (b) for at least one useful trait, wherein aportion of the population of progeny plants express MSH1; and, d).selecting a progeny plant comprising the trait that expresses MSH1,wherein the trait is associated with one or more mutated chromosomalloci, are provided. In certain embodiments, the mutated chromosomal locicomprise nucleotide inversions, insertions, deletions, substitutions, orcombinations thereof. In certain embodiments, the chromosomal locicomprise mutations are reversible. In certain embodiments, thechromosomal loci comprise mutations are irreversible. In certainembodiments of any of the preceding methods, the method furthercomprises the step of producing seed from: i) a selfed progeny plant ofstep (d), ii) an out-crossed progeny plant of step (d), or, iii) fromboth of a selfed and an out-crossed progeny plant of step (d). Incertain embodiments, the methods can further comprise the step ofassaying seed or plants grown from the seed for the presence of thetrait. In certain embodiments of any of the preceding methods, the firstparental plant or plant cell comprises a transgene that can suppressexpression of MSH1. In certain embodiments of the methods, the transgeneis selected from the group of transgenes that suppress expression ofMSH1 by producing a small inhibitory RNA (siRNA), a microRNA (miRNA), aco-suppressing sense RNA, and/or an anti-sense RNA. In certainembodiments of any of the preceding methods, the first parental plant orplant cell can be obtained by crossing a female plant with a distinctmale plant, wherein at least one of the female or male plants comprise atransgene that suppresses expression of the endogenous MSH1 gene of theparental plant(s), and wherein the plants were isogenic inbred linesprior to introduction of the transgene. In certain embodiments of any ofthe preceding methods, the first parental plant or plant cell wasisogenic to the second parental plant prior to suppression of MSH1 inthe first parental plant or plant cell. In certain embodiments of any ofthe preceding methods the trait is selected from the group consisting ofyield, male sterility, non-flowering, resistance to biotic stress, andresistance to abiotic stress. In certain embodiments, abiotic stress canbe selected from the group consisting of drought stress, osmotic stress,nitrogen stress, phosphorous stress, mineral stress, heat stress, coldstress, and/or light stress. In certain embodiments, resistance toabiotic stress can include drought tolerance, high light tolerance, heattolerance, cold tolerance, and salt tolerance. In certain embodiments ofthe methods, biotic stress can be selected from the group consisting ofplant fungal pathogens, plant bacterial pathogens, plant viralpathogens, insects, nematodes, and herbivores, and any combinationthereof. In certain embodiments of any of the preceding methods, thetrait is not caused by substoichiometric shifting (SSS) in mitochondriaof the progeny plant. In certain embodiments of any of the precedingmethods, the trait is male sterility and is not caused bysubstoichiometric shifting (SSS) in mitochondria of the progeny plant.In certain embodiments of any of the preceding methods, the progenyplant in step (d) or progeny thereof exhibit an improvement in the traitin comparison to a plant that had not been subjected to suppression ofMSH1 expression but was otherwise isogenic to the first parental plantor plant cell parental plants. In certain embodiments of any of thepreceding methods, the plant is a crop plant. In certain embodiments ofany of the preceding methods, the crop plant is selected from the groupconsisting of cotton, canola, wheat, barley, flax, oat, rye, turf grass,sugarcane, alfalfa, banana, broccoli, cabbage, carrot, cassava,cauliflower, celery, citrus, a cucurbit, eucalyptus, garlic, grape,onion, lettuce, pea, peanut, pepper, potato, poplar, pine, sunflower,safflower, soybean, strawberry, sugar beet, sweet potato, tobacco,cassava, cauliflower, celery, citrus, cotton, a cucurbit, eucalyptus,garlic, grape, onion, lettuce, pea, peanut, pepper, potato, poplar,pine, sunflower, safflower, strawberry, sugar beet, sweet potato,tobacco, cassava, cauliflower, celery, citrus, cucurbits, eucalyptus,garlic, grape, onion, lettuce, pea, peanut, pepper, poplar, pine,sunflower, safflower, soybean, strawberry, sugar beet, tobacco,Jatropha, Camelina, and Agave. In certain embodiments of any of thepreceding methods, the crop plant is selected from the group consistingof corn, soybean, cotton, canola, wheat, rice, tomato, tobacco, millet,and sorghum. In certain embodiments of any of the preceding methods, thecrop is sorghum. In certain embodiments of any of the preceding methods,the crop is sorghum and the trait is selected from the group consistingof panicle length, panicle weight, dry biomass, and combinationsthereof.

Also provided herein are plants, plant parts including seeds, orproducts of the plants or seeds, that exhibit useful traits caused byalterations and/or mutations in chromosomal loci resulting fromsuppression of MSH1. In certain embodiments, the plant seed, or productsthereof that exhibit useful traits caused by alterations and/ormutations in chromosomal loci resulting from suppression of MSH1exhibits an improvement in at least one useful trait in comparison to aplant, plant parts including seeds, or products of the plants or seeds,that had not been subjected to suppression of MSH1 expression but wasotherwise isogenic to the first parental plant or plant cell. In certainembodiments, such plants, seeds or products of the invention thatexhibit useful traits caused by alterations and/or mutations inchromosomal loci resulting from suppression of MSH1 can comprise one ormore alterations and/or mutations in one or more chromosomal loci thatwere induced by MSH1 suppression. In certain embodiments, a plant or acrop plant produced by any of the preceding methods, wherein the cropplant exhibits an improvement in at least one useful trait in comparisonto a plant that had not been subjected to suppression of MSH1 expressionbut was otherwise isogenic to the first parental plant or plant cell isprovided. In certain embodiments, any of the aforementioned plants orcrop plants is inbred and exhibits an improvement in at least one usefultrait in comparison to the parental plant or parental plants. Alsoprovided herein are seed obtained from any of the aforementioned plantsor crop plants. Also provided herein are processed products from any ofthe aforementioned plants, crop plants or seeds, wherein the productcomprises a detectable amount of a chromosomal DNA, a mitochondrial DNA,a plastid DNA, plastid and mitochondrial DNA, or any combinationthereof. In certain embodiments, the product can comprises a detectableamount of a chromosomal DNA that comprise one or more alterations and/ormutations in one or more chromosomal loci that were induced by MSH1suppression. In certain embodiments of any of the aforementionedprocessed products, the product can be oil, meal, lint, hulls, or apressed cake.

Also provided herein are methods for producing seed that compriseharvesting seed from any of the aforementioned plants or crop plants ofthe invention. In certain embodiments, methods for producing a lot ofseed comprising the steps of selfing a population of plants or cropplants of the invention, growing the selfed plants, and harvesting seedtherefrom are provided. In certain embodiments, the harvested seed or aplant obtained therefrom exhibits the improvement in at least one usefultrait.

Also provided herewith are methods of using any of the aforementionedplants or crop plants of the invention that comprise any of the improvedtraits, where the methods comprise growing, propagating, or cultivatingthe plants or crop plants of the invention that exhibit the improvedtrait. Methods of obtaining improved yields that comprise harvesting anyplant part including a seed of any of the aforementioned plants or cropplants of the invention are also provided. In certain embodiments, theharvested seed or a plant obtained therefrom exhibits the improvement inat least one useful trait.

In certain embodiments, methods for identifying one or more alteredchromosomal loci in a plant that can confer a useful trait are provided.In one embodiment, methods comprising the steps of: a. comparing one ormore chromosomal regions in a reference plant that does not exhibit theuseful trait to one or more corresponding chromosomal regions in a testplant that does exhibit the useful trait, wherein the test plantexpresses MSH1 and was obtained from a parental plant or plant cellwherein MSH1 had been suppressed; and, b. selecting for one or morealtered chromosomal loci present in the test plant that are absent inthe reference plant and that are associated with the useful trait areprovided. In certain embodiments, an altered chromosomal locus comprisesa chromosomal DNA methylation state, a post-translation modification ofa histone protein associated with a chromosomal locus, or anycombination thereof. In certain embodiments, the selection comprisesisolating a plant or progeny plant comprising the altered chromosomallocus or obtaining a nucleic acid associated with the alteredchromosomal locus. In certain embodiments, both the reference plant andthe test plant are obtained from a population of progeny plants obtainedfrom a parental plant or plant cell wherein MSH1 had been suppressed. Incertain embodiments, both the reference plant and the parental plant orplant cell were isogenic prior to suppression of MSH1 in the parentalplant or plant cell. In certain embodiments, the useful trait isselected from the group consisting of yield, male sterility,non-flowering, biotic stress resistance, and abiotic stress resistance.In certain embodiments, abiotic stress can be selected from the groupconsisting of drought stress, osmotic stress, nitrogen stress,phosphorous stress, mineral stress, heat stress, cold stress, and/orlight stress. In certain embodiments, resistance to abiotic stress caninclude drought tolerance, high light tolerance, heat tolerance, coldtolerance, and salt tolerance. In certain embodiments of the methods,the biotic stress resistance can be selected from the group consistingof plant fungal pathogen resistance, plant bacterial pathogenresistance, plant viral pathogen resistance, insect resistance, nematoderesistance, and herbivore resistance, and any combination thereof. Incertain embodiments, the useful trait is selected from the groupconsisting of enhanced lodging resistance, enhanced growth rate,enhanced biomass, enhanced tillering, enhanced branching, delayedflowering time, and delayed senescence. Also provided herein are alteredchromosomal loci identified by any of the preceding methods. Suchaltered chromosomal loci can comprise a chromosomal DNA methylationstate, a post-translation modification of a histone protein associatedwith a chromosomal locus, or any combination thereof.

Also provided herein are plants comprising any of the alteredchromosomal loci identified by any of the preceding methods.

Also provided herein are methods for producing a plant exhibiting auseful trait. In certain embodiments, these methods can comprise thesteps of: a. introducing a chromosomal modification associated with auseful trait into a plant, wherein the chromosomal modificationcomprises an altered chromosomal locus induced by MSH1 suppressionassociated with the useful trait, a transgene that provides for the samegenetic effect as an altered chromosomal locus induced by MSH1suppression associated with the useful trait, or a chromosomal mutationthat provides for the same genetic effect as an altered chromosomallocus induced by MSH1 suppression associated with the useful trait; and,b. selecting for a plant that comprises the chromosomal modification andexhibits the useful trait. In certain embodiments, the methods canfurther comprise the step of producing seed from: i) a selfed progenyplant of the selected plant of step (b), ii) an out-crossed progenyplant of the selected plant of step (b), or, iii) from both of a selfedand an out-crossed progeny plant of the selected plant of step (b). Incertain embodiments of the methods, the chromosomal modification cancomprise an altered chromosomal locus and the plant is selected byassaying for the presence of a chromosomal DNA methylation state, apost-translation modification of a histone protein associated with achromosomal locus, or any combination thereof, that is associated withthe altered chromosomal locus. In certain embodiments, the chromosomalmodification comprises the transgene or the chromosomal mutation and theplant is selected by assaying for the presence of the transgene or thechromosomal mutation. In other embodiments, the plant is selected byassaying for the presence of the useful trait. In certain embodiments,the chromosomal modification comprises an altered chromosomal locus andthe altered chromosomal locus comprises a chromosomal DNA methylationstate, a post-translation modification of a histone protein associatedwith a chromosomal locus, or any combination thereof. In certainembodiments, the altered chromosomal locus has a genetic effect thatcomprises a reduction in expression of a gene and the chromosomalmodification comprises a transgene or a chromosomal mutation thatprovides for a reduction in expression of the gene. In certainembodiments where the altered chromosomal locus has a genetic effectthat comprises a reduction in expression of a gene and the chromosomalmodification comprises a transgene, the transgene reduces expression ofthe gene by producing a small inhibitory RNA (siRNA), a microRNA(miRNA), a co-suppressing sense RNA, and/or an anti-sense RNA directedto the gene. In certain embodiments, the altered chromosomal locus has agenetic effect that comprises an increase in expression of a gene andthe chromosomal modification comprises a transgene or a chromosomalmutation that provides for an increase in expression of the gene. Incertain embodiments of any of the preceding methods, the useful trait isselected from the group consisting of yield, male sterility,non-flowering, biotic stress resistance, and abiotic stress resistance.In certain embodiments, abiotic stress can be selected from the groupconsisting of drought stress, osmotic stress, nitrogen stress,phosphorous stress, mineral stress, heat stress, cold stress, and/orlight stress. In certain embodiments, resistance to abiotic stress caninclude drought tolerance, high light tolerance, heat tolerance, coldtolerance, and salt tolerance. In certain embodiments of the methods,biotic stress can be selected from the group consisting of plant fungalpathogens, plant bacterial pathogens, plant viral pathogens, insects,nematodes, and herbivores, and any combination thereof. In certainembodiments of the methods, the useful trait is selected from the groupconsisting of enhanced lodging resistance, enhanced growth rate,enhanced biomass, enhanced tillering, enhanced branching, delayedflowering time, and delayed senescence. Also provided herein are plantsmade by any of the preceding methods. In certain embodiments of any ofthe preceding methods, the plant is a crop plant. In certain embodimentsof any of the preceding methods, the crop plant is selected from thegroup consisting of cotton, canola, wheat, barley, flax, oat, rye, turfgrass, sugarcane, alfalfa, banana, broccoli, cabbage, carrot, cassava,cauliflower, celery, citrus, a cucurbit, eucalyptus, garlic, grape,onion, lettuce, pea, peanut, pepper, potato, poplar, pine, sunflower,safflower, soybean, strawberry, sugar beet, sweet potato, tobacco,cassava, cauliflower, celery, citrus, cotton, a cucurbit, eucalyptus,garlic, grape, onion, lettuce, pea, peanut, pepper, potato, poplar,pine, sunflower, safflower, strawberry, sugar beet, sweet potato,tobacco, cassava, cauliflower, celery, citrus, cucurbits, eucalyptus,garlic, grape, onion, lettuce, pea, peanut, pepper, poplar, pine,sunflower, safflower, soybean, strawberry, sugar beet, tobacco,Jatropha, Camelina, and Agave. In certain embodiments of any of thepreceding methods, the crop plant is selected from the group consistingof corn, soybean, cotton, canola, wheat, rice, tomato, tobacco, millet,and sorghum. In certain embodiments of any of the preceding methods, thecrop is sorghum. In certain embodiments of any of the preceding methods,the crop is sorghum and the trait is selected from the group consistingof panicle length, panicle weight, dry biomass, and combinationsthereof.

Also provided herein are plants, plant parts, including but not limitedto, seeds, leaves, stems roots, and flowers, or products of the plants,or plant parts including but not limited to seeds, that comprise achromosomal modification associated with a useful trait or a chromosomalalteration associated with a useful trait. In certain embodiments, theplant part can comprise a non-regenerable plant part or non-regenerableportion of a plant part. In certain embodiments, the products can beprocessed products that include, but are not limited to, a feed or ameal obtained from a plant part. In certain embodiments, the plantsseed, or products thereof that exhibit useful traits caused by achromosomal modification exhibits an improvement in at least one usefultrait in comparison to a plant, plant parts including seeds, or productsof the plants or seeds, that do not comprise the chromosomalmodification. In certain embodiments, such plants, seeds or productsthat that exhibit useful traits, can comprise a chromosomal modificationthat comprises a altered chromosomal locus induced by MSH1 suppressionassociated with the useful trait, a transgene that provides for the samegenetic effect as an altered chromosomal locus induced by MSH1suppression associated with the useful trait, or a chromosomal mutationthat provides for the same genetic effect as an altered chromosomallocus induced by MSH1 suppression associated with the useful trait. Incertain embodiments, such plants, plant parts, seeds or products thatexhibit useful traits can comprise an altered chromosomal locus thatcomprises a chromosomal DNA methylation state, a post-translationmodification of a histone protein associated with a chromosomal locus,or any combination thereof. In certain embodiments, the alteredchromosomal locus that comprises a chromosomal DNA methylation state cancomprise a distinguishing portion of the altered chromosomal locus thatis not found in plants, plant parts, or plant products that have notbeen subject to MSH1 supression. In certain embodiments, thedistinguishing portion of the altered chromosomal locus can comprise amethylated DNA molecule of at least about 25 nucleotides, 50nucleotides, 100 nucleotides, 200 nucleotides, 500 nucleotides, or more.In certain embodiments, a plant, plant cell, or plant product producedby any of the preceding methods, wherein the plant exhibits animprovement in at least one useful trait in comparison to a plant thatdoes not comprise the chromosomal alteration but was otherwise isogenicto the first parental plant or plant cell is provided. In certainembodiments, any of the aforementioned plants is inbred and exhibits animprovement in at least one useful trait in comparison to the parentalplant or parental plants. Also provided herein are seed obtained fromany of the aforementioned plants, plant cells, or crop plants. Alsoprovided herein are processed products from any of the aforementionedplants, crop plants or plant parts including, but not limited to seeds,wherein the product comprises a detectable amount of a chromosomal DNAcomprising any of the aforementioned chromosomal modifications thatinclude, but are not limited to, an altered chromosomal locus, atransgene that provides for the same genetic effect as an alteredchromosomal locus induced by MSH1 suppression associated with the usefultrait, or a chromosomal mutation that provides for the same geneticeffect as an altered chromosomal locus induced by MSH1 suppressionassociated with the useful trait. In certain embodiments of any of theaforementioned processed products, the product can be oil, meal, lint,hulls, or a pressed cake.

Also provided herein are methods for producing seed that compriseharvesting seed from any of the aforementioned plants or crop plants ofthe invention. In certain embodiments, methods for producing a lot ofseed comprising the steps of selfing a population of plants or cropplants of the invention, growing the selfed plants, and harvesting seedtherefrom are provided.

Also provided herewith are methods of using any of the aforementionedplants or crop plants of the invention that comprise any of the improvedtraits, where the methods comprise growing, propagating, or cultivatingthe plants or crop plants of the invention that exhibit the improvedtrait. Methods of obtaining improved yields that comprise harvesting anyplant part including a seed of any of the aforementioned plants or cropplants of the invention are also provided.

Use in any process of any of the plants, plant parts or portions thereofincluding but not limited to plant cells, non-regenerable plant parts orportions thereof including but not limited to plant cells, or processedplant products is also provided herein. Processes for which the plants,plant parts or portions thereof, non-regenerable plant parts or portionsthereof, or processed plant products provided herein can be usedinclude, but are not limited to, use in breeding, use as biofuel, use asanimal feed, use in human food products, and use in any industrial,food, or feed manufacturing processes.

Also provided herein are seed that exhibit the useful trait(s) andplants obtained from the seed that exhibit the improvement in the usefultrait(s). In certain embodiments, the seed can comprise an alteredchromosomal loci that is associated with the useful trait(s) or thatimpart the useful trait(s).

In certain embodiments, the plants, plant parts, non-regenerable plantparts, plant cells, non-regenerable plant cells, plant products orprocessed plant product provided herein can comprise a detectable amountof a chromosomal DNA that comprises an altered chromosomal locus inducedby MSH1 suppression associated with the useful trait, a transgene thatprovides for the same genetic effect as an altered chromosomal locusinduced by MSH1 suppression associated with the useful trait, or achromosomal mutation that provides for the same genetic effect as analtered chromosomal locus induced by MSH1 suppression associated withthe useful trait. In certain embodiments, the altered chromosomal locusthat comprises a chromosomal DNA methylation state can comprise adistinguishing portion of the altered chromosomal locus that is notfound in plants, plant cells, non-regenerable plant cells, plant parts,non-regenerable plant parts, plant products, or processed plant productsthat have not been subject to MSH1 suppression. In certain embodiments,the distinguishing portion of the altered chromosomal locus can comprisea methylated DNA molecule of at least about 25 nucleotides, 50nucleotides, 100 nucleotides, 200 nucleotides, 500 nucleotides, or more.Processed products provided herein comprising the chromosomal DNA ordistinguishing portions thereof include, but are not limited to,products that comprise oil, meal, lint, hulls, or a pressed cake.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate certain embodiments of the presentinvention. In the drawings:

FIG. 1 illustrates various phenotypes that are observed in variousplants subjected to MSH1 suppression such as cytoplasmic male sterility,variegation and altered chloroplast development, reduced growth rate anddwarfing, altered flowering time or non-flowering, reduced flavonoidbiosynthesis and lack of anthocyanins, enhanced pathogen susceptibility,altered leaf morphologies, and high light tolerance.

FIG. 2 illustrates leaf variegation in Arabidopsis (top), tomato(middle), and sorghum (bottom panel) plants that had been subjected toMSH1 suppression.

FIG. 3 illustrates dwarfing in Sorghum (top) and tomato (bottom panel)plants that had been subjected to MSH1 suppression.

FIG. 4 illustrates mitochondrial DNA rearrangements in Arabidopsis thathad been subjected to MSH1 suppression.

FIG. 5 illustrates increases in Reactive Oxygen Species (ROS) that areobserved in tomato, tobacco, and millet plants subjected to MSH1suppression.

FIG. 6 illustrates an exemplary and non-limiting scheme for obtainingplants that exhibit various types of heritable phenotypic variationreferred to herein as “discrete variation” (V_(D)) as a result of havingbeen subjected to MSH1 suppression and for obtaining plant lines thatcan exhibit “quantitative variation” or “V_(Q)” and various usefultraits.

FIG. 7 illustrates an Arabidopsis plant line (msh1×Col-0 F₃) thatexhibits increases in biomass relative to an otherwise isogenic parentalplant that had not been subjected to MSH1 suppression (Col-0).

FIG. 8 illustrates the distribution of plant heights (in cM) that areobtained in distinct sorghum lines GAII-11 (squares), GA11-15(triangles), GAII-22 (opposing brackets), GAII-24, and GAII-28 (circles)derived from outcrosses of plants where MSH1 expression was suppressed.The wild type reference line is fx WT (diamonds).

FIG. 9 illustrates the distribution of panicle weights (in grams) thatare obtained in distinct sorghum lines GAII-11 (squares), GA11-15(triangles), GAII-22 (opposing brackets), GAII-24, and GAII-28 (circles)derived from outcrosses of plants where MSH1 expression was suppressed.The wild type reference line is fx WT (diamonds).

FIG. 10 illustrates the distribution of grain yield (in grams) that areobtained in distinct sorghum lines GAII-11 (squares), GA11-15(triangles), GAII-22 (opposing brackets), GAII-24, and GAII-28 (circles)derived from outcrosses of plants where MSH1 expression was suppressed.The wild type reference line is fx WT (diamonds).

FIG. 11A-H illustrates the enhanced growth phenotype of MSH1-epi linesin Arabidopsis and sorghum. The transgene and crossing procedures usedto derive sorghum and Arabidopsis epi-populations are indicated. (A) Thephenotype of the F1 progeny derived from crossing Tx430×MSH1-dr. (B)Field grown epiF2, F3 and F4 sorghum lines show variation in plantarchitecture and height. (C) Panicles from Tx430 (on left, 66 gm, 8 mmstem) versus epi-F2 individual (on right, 112 gm, 11 mm stem). (D) seedyield from the panicles shown in C. (E) The MSH1-dr sorghum phenotypeunder field conditions. (F) Evidence of enhanced rosette growth in anepi-F4 line of Arabidopsis. (G) Arabidopsis epi-F4 plants shown enhancedplant biomass, rosette diameter and flower stem diameter relative toCol-0. Data shown as mean±SE from >6. (H) The Arabidopsis epiF4phenotype at flowering.

FIG. 12 illustrates enhanced phenotypic variation in sorghum MSH1-epiF2lines. Phenotypic distributions are shown for plant height and grainyield from three independent sorghum epiF2 populations grown in twofield plantings. Population means are shown by dashed vertical lines.

FIG. 13A, B illustrate phenotypic variation in sorghum MSH1-epiF2, F3and F4 lines. (A) MSH1-epiF4 lines selected for plant height and grainyield per panicle. For plant height, lines 4b-10, 10.3 and 3a.2 wereselected for low plant height, all others were selected for tall. Forgrain yield, line 15.2 was selected for low yield, all others wereselected for high. (B) Box plots showing individual population responseto selection for four independent populations. Horizontal dashed linerepresents mean for Tx430 wildtype. In the case of grain yield, F3selection was carried out in the greenhouse.

FIG. 14A-E illustrates that MSH1-epi enhanced growth in Arabidopsis isassociated with chloroplast effects. (A) Mitochondrialhemi-complementation line AOX-MSH1×Col-0 F1; (B) Plastid-complementedSSU-MSH1×Col-0 F2 appears identical to Col-0 wildtype, (C) Rosettediameter and fresh biomass of SSU-MSH1-derived F2 lines relative toCol-0; (D) Mitochondrial-complemented AOX-MSH1×Col-0 F2 showing enhancedgrowth; (E) Rosette diameter and fresh biomass of AOX-MSH1-derived F2lines is significantly greater (P<0.05) than Col-0. (F) Enhanced growthphenotype in the F4 generation of A0X-MSH1×Col-0.

FIG. 15A-C illustrates Genome-wide 5-methyl-cytosine patterns inArabidopsis Col-0 wildtype and MSH1-epiF3 lines. (A) Relativecontributions of CG, CHG and CHH methylation to differential andnon-differential methylation of the genome. Note that the intergenicregion is at the top of the bar, followed in order by TE gene,pseudogenes, ncRNA, 3′-UTR, 5′-UTR, intron, and CDS. (B) Distribution ofCG-DMPs and CG-N-DMPs along each chromosome, with data normalized to thehighest value for each chromosome in parallel to the analysis procedureused by Becker et al. Nature 480, 245 (2011). (C) Col-0 methylationanalysis taken from FIG. 1 c in Becker et al. (Ibid) to demonstrate thesimilarity of NDMP patterns and the dissimilarity of DMP.

FIG. 16 illustrates Arabidopsis F1 plants resulting from crosses of themsh1 chloroplast hemi-complementation line×Col-0 wildtype.Transgene-mediated chloroplast hemicomplementation of msh1 restores thewildtype phenotype. However, crosses of these hemicomplemented lines toCol-0 results in ca. 25% of the plants displaying leaf curl to varyingintensities in the F1. The cause of this phenotype is not yet known, butit is no longer visible in derived F2 populations.

FIG. 17 illustrates the distribution of flowering time in ArabidopsisCol-0, epiF4 and epiF5 lines. Each distribution is plotted based on aminimum of 50 plants.

FIG. 18A, B, C illustrates the validation of differentially methylatedregions between arabidopsis lines col-0 and msh1-epif3 using bisulfatesequencing. Alignment of DMR region within AT3G27150 (Target gene ofMIR2111-5p). Highlighted Gs (i.e. underlined in the figure) arepredicted to be unmethylated in Col-0 and methylated in MSH1-epiF3. Thesequences of FIG. 18A, B, and C are provided in the sequence listing asfollows: AT3G27150 (SEQ ID NO:27), Col0-MIR2-2 (SEQ ID NO:28),Col0-MIR2-3 (SEQ ID NO:29), Col0-MIR2-4 (SEQ ID NO:30), Col0-MIR2-5 (SEQID NO:31), Col0-MIR2-6 (SEQ ID NO:32), Col0-MIR2-10 (SEQ ID NO:33),Col0-MIR2-11 (SEQ ID NO:34), Col0-MIR2-12 (SEQ ID NO:35), Col0-MIR2-26(SEQ ID NO:36), Col0-MIR2-27 (SEQ ID NO:37), Col0-MIR2-28 (SEQ IDNO:38), Col0-MIR2-29 (SEQ ID NO:39), F3-Mir2-1 (SEQ ID NO:40), F3-Mir2-2(SEQ ID NO:41), F3-Mir2-4 (SEQ ID NO:42), F3-Mir2-5 (SEQ ID NO:43),F3-Mir2-7 (SEQ ID NO:44), F3-Mir2-11 (SEQ ID NO:45), F3-Mir2-12 (SEQ IDNO:46), F3-Mir2-15 (SEQ ID NO:47), F3-Mir2-16 (SEQ ID NO:48), F3-Mir2-27(SEQ ID NO:49), and F3-Mir2-28 (SEQ ID NO:50).

DETAILED DESCRIPTION I. Definitions

As used herein, the phrase “chromosomal modification” refers to any of:a) an “altered chromosomal loci” and an “altered chromosomal locus”; b)“mutated chromosomal loci”, a “mutated chromosomal locus”, “chromosomalmutations” and a “chromosomal mutation”; or c) a transgene.

As used herein, the phrases “altered chromosomal loci” (plural) or“altered chromosomal locus (singular) refer to portions of a chromosomethat have undergone a heritable and reversible epigenetic changerelative to the corresponding parental chromosomal loci. Heritable andreversible genetic changes in altered chromosomal loci include, but arenot limited to, methylation of chromosomal DNA, and in particular,methylation of cytosine residues to 5-methylcytosine residues, and/orpost-translational modification of histone proteins, and in particular,histone modifications that include, but are not limited to, acetylation,methylation, ubiquitinylation, phosphorylation, and sumoylation(covalent attachment of small ubiquitin-like modifier proteins). As usedherein, “chromosomal loci” refer to loci in chromosomes located in thenucleus of a cell.

As used herein, the term “comprising” means “including but not limitedto”. As used herein, the phrases “mutated chromosomal loci” (plural)(plural), “mutated chromosomal locus” (singular), “chromosomalmutations” and “chromosomal mutation” refer to portions of a chromosomethat have undergone a heritable genetic change in a nucleotide sequencerelative to the nucleotide sequence in the corresponding parentalchromosomal loci. Mutated chromosomal loci comprise mutations thatinclude, but are not limited to, nucleotide sequence inversions,insertions, deletions, substitutions, or combinations thereof. Incertain embodiments, the mutated chromosomal loci can comprise mutationsthat are reversible. In this context, reversible mutations in thechromosome can include, but are not limited to, insertions oftransposable elements, defective transposable elements, and certaininversions. In certain embodiments, the chromosomal loci comprisemutations are irreversible. In this context, irreversible mutations inthe chromosome can include, but are not limited to, deletions.

As used herein, the term “discrete variation” or “V_(D)” refers todistinct, heritable phenotypic variation that includes traits of malesterility, dwarfing, variegation, and/or delayed flowering time that canbe observed either in any combination or in isolation.

As used herein, the term “MSH-dr” refers to changes in plant tillering,height, internode elongation and stomatal density that are observed inplants subjected to MSH1 suppression.

As used herein, the phrase “quantitative variation” or “V_(Q)” refers tophenotypic variation that is observed in individual progeny linesderived from outcrosses of plants where MSH1 expression was suppressedand that exhibit discrete variation to other plants.

As used herein the terms “microRNA” or “miRNA” refers to both a miRNAthat is substantially similar to a native miRNA that occurs in a plantas well as to an artificial miRNA. In certain embodiments, a transgenecan be used to produce either a miRNA that is substantially similar to anative miRNA that occurs in a plant or an artificial miRNA.

As used herein, the phrase “obtaining a nucleic acid associated with thealtered chromosomal locus” refers to any method that provides for thephysical separation or enrichment of the nucleic acid associated withthe altered chromosomal locus from covalently linked nucleic that hasnot been altered. In this context, the nucleic acid does not necessarilycomprise the alteration (i.e. such as methylation) but at leastcomprises one or more of the nucleotide base or bases that are altered.Nucleic acids associated with an altered chromosomal locus can thus beobtained by methods including, but not limited to, molecular cloning,PCR, or direct synthesis based on sequence data.

The phrase “operably linked” as used herein refers to the joining ofnucleic acid sequences such that one sequence can provide a requiredfunction to a linked sequence. In the context of a promoter, “operablylinked” means that the promoter is connected to a sequence of interestsuch that the transcription of that sequence of interest is controlledand regulated by that promoter. When the sequence of interest encodes aprotein and when expression of that protein is desired, “operablylinked” means that the promoter is linked to the sequence in such a waythat the resulting transcript will be efficiently translated. If thelinkage of the promoter to the coding sequence is a transcriptionalfusion and expression of the encoded protein is desired, the linkage ismade so that the first translational initiation codon in the resultingtranscript is the initiation codon of the coding sequence.Alternatively, if the linkage of the promoter to the coding sequence isa translational fusion and expression of the encoded protein is desired,the linkage is made so that the first translational initiation codoncontained in the 5′ untranslated sequence associated with the promoteris linked such that the resulting translation product is in frame withthe translational open reading frame that encodes the protein desired.Nucleic acid sequences that can be operably linked include, but are notlimited to, sequences that provide gene expression functions (i.e., geneexpression elements such as promoters, 5′ untranslated regions, introns,protein coding regions, 3′ untranslated regions, polyadenylation sites,and/or transcriptional terminators), sequences that provide DNA transferand/or integration functions (i.e., site specific recombinaserecognition sites, integrase recognition sites), sequences that providefor selective functions (i.e., antibiotic resistance markers,biosynthetic genes), sequences that provide scoreable marker functions(i.e., reporter genes), sequences that facilitate in vitro or in vivomanipulations of the sequences (i.e., polylinker sequences, sitespecific recombination sequences, homologous recombination sequences),and sequences that provide replication functions (i.e., bacterialorigins of replication, autonomous replication sequences, centromericsequences).

As used herein, the phrase “suppressing expression of MSH1 gene(s)”refers to any genetic or environmental manipulation that provides fordecreased levels of functional MSH1 activity in a plant or plant cellrelative to the levels of functional MSH1 activity that occur in anotherwise isogenic plant or plant cell that had not been subjected tothis genetic or environmental manipulation.

As used herein, the term “transgene”, in the context of a chromosomalmodification, refers to any DNA from a heterologous source that has beenintegrated into a chromosome that is stably maintained in a host cell.In this context, heterologous sources for the DNA include, but are notlimited to, DNAs from an organism distinct from the host cell organism,species distinct from the host cell species, varieties of the samespecies that are either distinct varieties or identical varieties, DNAthat has been subjected to any in vitro modification, recombinant DNA,and any combination thereof.

As used herein, the term “non-regenerable” refers to a plant part orplant cell that can not give rise to a whole plant.

II. Description Overview

Methods for introducing heritable and epigenetic and/or geneticvariation that result in plants that exhibit useful traits are providedherewith along with plants, plant seeds, plant parts, plant cells, andprocessed plant products obtainable by these methods. In certainembodiments, methods provided herewith can be used to introduceepigenetic and/or genetic variation into varietal or non-hybrid plantsthat result in useful traits as well as useful plants, plant partsincluding, but not limited to, seeds, plant cells, and processed plantproducts that exhibit, carry, or otherwise reflect benefits conferred bythe useful traits. In other embodiments, methods provided herewith canbe used to introduce epigenetic and/or genetic variation into plantsthat are also amenable to hybridization.

In most embodiments, methods provided herewith involve suppressingexpression of the plant MSH1 gene, restoring expression of a functionalplant MSH1 gene, and selecting progeny plants that exhibit one or moreuseful traits. In certain embodiments, these useful traits areassociated with either one or more altered chromosomal loci that haveundergone a heritable and reversible epigenetic change, with one or moremutated chromosomal loci that have undergone a heritable genetic change,or combinations thereof.

III. Suppression of MSH1 Expression in Plants or Plant Cells.

In general, methods provided herewith for introducing epigenetic and/orgenetic variation plants simply require that MSH1 expression besuppressed for a time sufficient to introduce the variation. As such, awide variety of MSH 1 suppression methods can be employed to practicethe methods provided herewith and the methods are not limited to aparticular suppression technique.

Since both the MSH1 gene and the effects of MSH1 gene depletion appearto be highly conserved in plants, it is further anticipated that themethods provided herein can be applied to a variety of different plantsor plant cells. Sequences of MSH1 genes or fragments thereof fromArabidopsis, soybean, Zea mays, Sorghum, rice, Brachypodium, Vitisvinifera, cotton, and cucumber are provided herewith. In certainembodiments, such genes may be used directly in either the homologous ora heterologous plant species to provide for suppression of theendogenous MSH1 gene in either the homologous or heterologous plantspecies. A non-limiting, exemplary demonstration where a MSH1 gene fromone species was shown to be effective in suppressing the endogenous MSH1gene in both a homologous and a heterologous species is provided bySandhu et al. 2007, where a transgene that provides for an MSH1inhibitory RNA (RNAi) with tomato MSH1 sequences was shown to inhibitthe endogenous MSH1 genes of both tomato and tobacco. A transgene thatprovides for an MSH1 inhibitory RNA (RNAi) with maize MSH1 sequences caninhibit the endogenous MSH1 genes of millet, sorghum, and maize. MSH1genes from other plants including, but not limited to, cotton, canola,wheat, barley, flax, oat, rye, turf grass, sugarcane, alfalfa, banana,broccoli, cabbage, carrot, cassava, cauliflower, celery, citrus, acucurbit, eucalyptus, garlic, grape, onion, lettuce, pea, peanut,pepper, potato, poplar, pine, sunflower, safflower, soybean, strawberry,sugar beet, sweet potato, tobacco, cassava, cauliflower, celery, citrus,cotton, a cucurbit, eucalyptus, garlic, grape, onion, lettuce, pea,peanut, pepper, potato, poplar, pine, sunflower, safflower, strawberry,sugar beet, sweet potato, tobacco, cassava, cauliflower, celery, citrus,cucurbits, eucalyptus, garlic, grape, onion, lettuce, pea, peanut,pepper, poplar, pine, sunflower, safflower, soybean, strawberry, sugarbeet, tobacco, Jatropha, Camelina, and Agave can be obtained by avariety of techniques and used to suppress expression of either thecorresponding MSH1 gene in those plants or the MSH1 gene in a distinctplant. Methods for obtaining MSH1 genes for various plants include, butare not limited to, techniques such as: i) searching amino acid and/ornucleotide sequence databases comprising sequences from the plantspecies to identify the MSH1 gene by sequence identity comparisons; ii)cloning the MSH1 gene by either PCR from genomic sequences or RT-PCRfrom expressed RNA; iii) cloning the MSH1 gene from a genomic or cDNAlibrary using PCR and/or hybridization based techniques; iv) cloning theMSH1 gene from an expression library where an antibody directed to theMSH1 protein is used to identify the MSH1 containing clone; v) cloningthe MSH1 gene by complementation of an msh1 mutant or MSH1 deficientplant; or vi) any combination of (i), (ii), (iii), (iv), and/or (v).Recovery of the MSH1 gene from the plant can be readily determined orconfirmed by constructing a plant transformation vector that providesfor suppression of the gene, transforming the plants with the vector,and determining if plants transformed with the vector exhibit thecharacteristic responses that are typically observed in various plantspecies when MSH1 expression is suppressed that include leafvariegation, cytoplasmic male sterility (CMS), a reduced growth-ratephenotype, delayed or non-flowering phenotype, and enhancedsusceptibility to pathogens.

In certain embodiments, MSH1 genes or fragments thereof used in themethods provided herein will have nucleotide sequences with at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% nucleotide sequenceidentity to one or more of the MSH1 genes or fragments thereof providedherein that include, but are not limited to, SEQ ID NO:1, SEQ ID NO:3-10, and SEQ ID NO:14. In certain embodiments, MSH1 genes or fragmentsthereof used in the methods provided herein encode MSH1 proteins orportions thereof will have amino acid sequences with at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity toone or more of the MSH1 proteins provided herein that include, but arenot limited to, SEQ ID NO:2, and the MSH1 proteins encoded by SEQ ID NO:3-10. In certain embodiments, MSH1 genes or fragments thereof used inthe methods provided herein will have nucleotide sequences with at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% nucleotide sequenceidentity to one or more of the MSH1 genes fragments thereof, orthologsthereof, or homologs thereof, provided herein that include, but are notlimited to, SEQ ID NO:51 and SEQ ID NO:52. In certain embodiments, MSH1genes or fragments thereof used in the methods provided herein encodeMSH1 proteins or portions thereof will have amino acid sequences with atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% amino acidsequence identity to one or more of the MSH1 proteins or MSH1 homologsprovided herein that include, but are not limited to, the proteinsencoded by SEQ ID NO:51 and SEQ ID NO:52. MSH1 genes from plants otherthan those provided herein can also be identified by the encoded DNAbinding (Domain I), ATPase (Domain V), and carboxy-terminal GIY-YIG typeendonuclease (Domain VI) domains that characterize many MSH1 genes(Abdelnoor et al. 2006). In this regard, it is anticipated that MSH 1nucleic acid fragments of 18 to 20 nucleotides, but more preferably 21nucleotides or more, can be used to effect suppression of the endogenousMSH1 gene. In certain embodiments, MSH1 nucleic acid fragments of atleast 18, 19, 20, or 21 nucleotides to about 50, 100, 200, 500, or morenucleotides can be used to effect suppression of the endogenous MSH1gene.

In certain embodiments, suppression of MSH1 in a plant is effected witha transgene. Transgenes that can be used to suppress expression of MSH1include, but are not limited to, transgenes that producedominant-negative mutants of MSH1, a small inhibitory RNA (siRNA), amicroRNA (miRNA), a co-suppressing sense RNA, and/or an anti-sense RNAthat provide for inhibition of the endogenous MSH 1 gene. US patentsincorporated herein by reference in their entireties that describesuppression of endogenous plant genes by transgenes include U.S. Pat.No. 7,109,393, U.S. Pat. No. 5,231,020 and U.S. Pat. No. 5,283,184(co-suppression methods); and U.S. Pat. No. 5,107,065 and U.S. Pat. No.5,759,829 (antisense methods). In certain embodiments, transgenesspecifically designed to produce double-stranded RNA (dsRNA) moleculeswith homology to the MSH1 gene can be used to decrease expression of theendogenous MSH1 gene. In such embodiments, the sense strand sequences ofthe dsRNA can be separated from the antisense sequences by a spacersequence, preferably one that promotes the formation of a dsRNA(double-stranded RNA) molecule. Examples of such spacer sequencesinclude, but are not limited to, those set forth in Wesley et al., PlantJ., 27(6):581-90 (2001), and Hamilton et al., Plant J., 15:737-746(1998). One exemplary and non-limiting vector that has been shown toprovide for suppression of MSH1 in tobacco and tomato has been describedby Sandhu et al., 2007 where an intron sequence separates the sense andantisense strands of the MSH1 sequence.

In certain embodiments, transgenes that provide for MSH1 suppression cancomprise regulated promoters that provide for either induction ordown-regulation of operably linked MSH1 inhibitory sequences. In thiscontext, MSH1 inhibitory sequences can include, but are not limited to,dominant-negative mutants of MSH1, a small inhibitory RNA (siRNA), amicroRNA (miRNA), a co-suppressing sense RNA, and/or an anti-sense RNAthat provide for inhibition of the endogenous MSH1 gene of a plant. Suchpromoters can provide for suppression of MSH1 during controlled timeperiods by either providing or withholding the inducer or downregulator. Inducible promoters include, but are not limited to, a PR-1apromoter (US Patent Application Publication Number 20020062502) or a GSTII promoter (WO 1990/008826 A1). In other embodiments, both atranscription factor that can be induced or repressed as well as apromoter recognized by that transcription factor and operably linked tothe MSH1 inhibitory sequences are provided. Such transcriptionfactor/promoter systems include, but are not limited to: i) RF2a acidicdomain-ecdysone receptor transcription factors/cognate promoters thatcan be induced by methoxyfenozide, tebufenozide, and other compounds (USPatent Application Publication Number 20070298499); ii) chimerictetracycline repressor transcription factors/cognate chimeric promotersthat can be repressed or de-repressed with tetracycline (Gatz, C., etal. (1992). Plant J. 2, 397-404), and the like.

In still other embodiments, transgenic plants are provided where thetransgene that provides for MSH1 suppression is flanked by sequencesthat provide for removal for the transgene. Such sequences include, butare not limited to, transposable element sequences that are acted on bya cognate transposase. Non-limiting examples of such systems that havebeen used in transgenic plants include the cre-lox and FLP-FRT systems.

MSH1 suppression can be readily identified or monitored by moleculartechniques. In certain embodiments where the endogenous MSH1 is intactbut its expression is inhibited, production or accumulation of the RNAencoding MSH1 can be monitored. Molecular methods for monitoring MSH1RNA expression levels include, but are not limited to, use ofsemi-quantitive or quantitative reverse transcriptase polymerase chainreaction (qRT-PCR) techniques. The use of semi-quantitive PCR techniquesto monitor MSH1 suppression resulting from RNAi mediated suppression ofMSH1 has been described (Sandhu et al. 2007). Various quantitativeRT-PCR procedures including, but not limited to, TaqMan™ reactions(Applied Biosystems, Foster City, Calif. US), use of Scorpion™ orMolecular Beacon™ probes, or any of the methods disclosed in Bustin, S.A. (Journal of Molecular Endocrinology (2002) 29, 23-39) can be used. Itis also possible to use other RNA quantitation techniques such asQuantitative Nucleic Acid Sequence Based Amplification (Q-NASBA™) or theInvader™ technology (Third Wave Technologies, Madison, Wis.).

In certain embodiments where MSH1 suppression is achieved by use of amutation in the endogenous MSH1 gene of a plant, the presence or absenceof that mutation in the genomic DNA can be readily determined by avariety of techniques. Certain techniques can also be used that providefor identification of the mutation in a hemizygous state (i.e. where onechromosome carries the mutated msh1 gene and the other chromosomecarries the wild type MSH1 gene). Mutations in MSH1 DNA sequences thatinclude insertions, deletions, nucleotide substitutions, andcombinations thereof can be detected by a variety of effective methodsincluding, but not limited to, those disclosed in U.S. Pat. Nos.5,468,613, 5,217,863; 5,210,015; 5,876,930; 6,030,787; 6,004,744;6,013,431; 5,595,890; 5,762,876; 5,945,283; 5,468,613; 6,090,558;5,800,944; 5,616,464; 7,312,039; 7,238,476; 7,297,485; 7,282,355;7,270,981 and 7,250,252 all of which are incorporated herein byreference in their entireties. For example, mutations can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,210,015 discloses detection of annealed oligonucleotides where a 5′labelled nucleotide that is not annealed is released by the 5′-3′exonuclease activity. U.S. Pat. No. 6,004,744 discloses detection of thepresence or absence of mutations in DNA through a DNA primer extensionreaction. U.S. Pat. No. 5,468,613 discloses allele specificoligonucleotide hybridizations where single or multiple nucleotidevariations in nucleic acid sequence can be detected by a process inwhich the sequence containing the nucleotide variation is amplified,affixed to a support and exposed to a labeled sequence-specificoligonucleotide probe. Mutations can also be detected by probe ligationmethods as disclosed in U.S. Pat. No. 5,800,944 where sequence ofinterest is amplified and hybridized to probes followed by ligation todetect a labeled part of the probe. U.S. Pat. Nos. 6,613,509 and6,503,710, and references found therein provide methods for identifyingmutations with mass spectroscopy. These various methods of identifyingmutations are intended to be exemplary rather than limiting as themethods of the present invention can be used in conjunction with anypolymorphism typing method to identify the presence of absence ofmutations in an MSH1 gene in genomic DNA samples. Furthermore, genomicDNA samples used can include, but are not limited to, genomic DNAisolated directly from a plant, cloned genomic DNA, or amplified genomicDNA.

Mutations in endogenous plant MSH1 genes can be obtained from a varietyof sources and by a variety of techniques. A homologous replacementsequence containing one or more loss of function mutations in the MSH1gene and homologous sequences at both ends of the double stranded breakcan provide for homologous recombination and substitution of theresident wild-type MSH1 sequence in the chromosome with a msh1replacement sequence with the loss of function mutation(s). Such loss offunction mutations include, but are not limited to, insertions,deletions, and substitutions of sequences within an MSH1 gene thatresult in either a complete loss of MSH1 function or a loss of MSH1function sufficient to elicit alterations (i.e. heritable and reversibleepigenetic changes) in other chromosomal loci or mutations in otherchromosomal loci. Loss-of-function mutations in MSH1 include, but arenot limited to, frameshift mutations, pre-mature translational stopcodon insertions, deletions of one or more functional domains thatinclude, but are not limited to, a DNA binding (Domain I), an ATPase(Domain V) domain, and/or a carboxy-terminal GIY-YIG type endonucleasedomain, and the like. Also provided herein are mutations analogous theArabidopsis msh1 mutation that are engineered into endogenous MSH1 plantgene to obtain similar effects. Methods for substituting endogenouschromosomal sequences by homologous double stranded break repair havebeen reported in tobacco and maize (Wright et al., Plant J. 44, 693,2005; D'Halluin, et al., Plant Biotech. J. 6:93, 2008). A homologousreplacement msh1 sequence (i.e. which provides a loss of functionmutation in an MSH1 sequence) can also be introduced into a targetednuclease cleavage site by non-homologous end joining or a combination ofnon-homologous end joining and homologous recombination (reviewed inPuchta, J. Exp. Bot. 56, 1, 2005; Wright et al., Plant J. 44, 693,2005). In certain embodiments, at least one site specific doublestranded break can be introduced into the endogenous MSH1 gene by ameganuclease. Genetic modification of meganucleases can provide formeganucleases that cut within a recognition sequence that exactlymatches or is closely related to specific endogenous MSH1 targetsequence (WO/06097853A1, WO/06097784A1, WO/04067736A2, U.S.20070117128A1). It is thus anticipated that one can select or design anuclease that will cut within a target MSH1 sequence. In otherembodiments, at least one site specific double stranded break can beintroduced in the endogenous MSH1 target sequence with a zinc fingernuclease. The use of engineered zinc finger nuclease to providehomologous recombination in plants has also been disclosed (WO03/080809, WO 05/014791, WO 07014275, WO 08/021207). In still otherembodiments, mutations in endogenous MSH1 genes can be identifiedthrough use of the TILLING technology (Targeting Induced Local Lesionsin Genomes) as described by Henikoff et al. where traditional chemicalmutagenesis would be followed by high-throughput screening to identifyplants comprising point mutations or other mutations in the endogenousMSH1 gene (Henikoff et al., Plant Physiol. 2004, 135:630-636).

In certain embodiments, MSH1 suppression can be effected by exposingwhole plants, or reproductive structures of plants, to stress conditionsthat result in suppression of endogenous MSH1 gene. Such stressconditions include, but are not limited to, high light stress, and heatstress. Exemplary and non-limiting high light stress conditions includecontinuous exposure to about 300 to about 1200 μmol photons/m2.s forabout 24 to about 120 hours. Exemplary and non-limiting heat stressconditions include continuous exposure to temperatures of about 32° C.to about 37° C. for about 2 hours to about 24 hours. Exemplary andnon-limiting heat, light, and other environmental stress conditions alsothat can provide for MSH1 suppression are also disclosed for heat(Shedge et al. 2010), high light stress (Xu et al. 2011) and otherenvironmental stress conditions (Hruz et al. 2008).

Methods where MSH1 suppression is effected in cultured plant cells arealso provided herein. In certain embodiments, MSH1 suppression can beeffected by culturing plant cells under stress conditions that result insuppression of endogenous MSH1 gene. Such stress conditions include, butare not limited to, high light stress. Exemplary and non-limiting highlight stress conditions include continuous exposure to about 300 toabout 1200 μmol photons/m2.s for about 24 to about 120 hours. Exemplaryand non-limiting heat stress conditions include continuous exposure totemperatures of about 32° C. to about 37° C. for about 2 hours to about24 hours. Exemplary and non-limiting heat, light, and otherenvironmental stress conditions also that can provide for MSH1suppression are also disclosed for heat (Shedge et al. 2010), high lightstress (Xu et al. 2011) and other environmental stress conditions (Hruzet al. 2008). In certain embodiments, MSH1 suppression is effected incultured plant cells by introducing a nucleic acid that provides forsuch suppression into the plant cells. Nucleic acids that can be used toprovide for suppression of MSH1 in cultured plant cells include, but arenot limited to, transgenes that produce a small inhibitory RNA (siRNA),a microRNA (miRNA), a co-suppressing sense RNA, and/or an anti-sense RNAdirected to the MSH1 gene. Nucleic acids that can be used to provide forsuppression of MSH1 in cultured plant cells include, but are not limitedto, a small inhibitory RNA (siRNA) or a microRNA (miRNA) directedagainst the endogenous MSH1 gene. RNA molecules that provide forinhibition of MSH1 can be introduced by electroporation. Introduction ofinhibitory RNAs to cultured plant cells to inhibit target genes can incertain embodiments be accomplished as disclosed in Vanitharani et al.(Proc Natl Acad Sci USA., 2003, 100(16):9632-6), Qi et al. (NucleicAcids Res. 2004 Dec. 15; 32(22):e179), or J. Cheon et al. (Microbiol.Biotechnol. (2009), 19(8), 781-786).

MSH1 suppression can also be readily identified or monitored bytraditional methods where plant phenotypes are observed. For example,MSH1 suppression can be identified or monitored by observing organellareffects that include leaf variegation, cytoplasmic male sterility (CMS),a reduced growth-rate phenotype, delayed or non-flowering phenotype,and/or enhanced susceptibility to pathogens. Phenotypes indicative ofMSH1 suppression in various plants are shown in FIGS. 1, 2, and 3. Thesephenotypes that are associated with MSH1 suppression are referred toherein as “discrete variation” (V_(D)). MSH1 suppression can alsoproduce changes in plant tillering, height, internode elongation andstomatal density (referred to herein as “MSH1-dr”) that can be used toidentify or monitor MSH1 suppression in plants. Other biochemical andmolecular traits can also be used to identify or monitor MSH1suppression in plants MSH1 suppression. Such molecular traits caninclude, but are not limited to, changes in expression of genes involvedin cell cycle regulation, Giberrellic acid catabolism, auxinbiosynthesis, auxin receptor expression, flower and vernalizationregulators (i.e. increased FLC and decreased SOC1 expression), as wellas increased miR156 and decreased miR172 levels. Such biochemical traitscan include, but are not limited to, up-regulation of most compounds ofthe TCA, NAD and carbohydrate metabolic pathways, down-regulation ofamino acid biosynthesis, depletion of sucrose in certain plants,increases in sugars or sugar alcohols in certain plants, as well asincreases in ascorbate, alphatocopherols, and stress-responsive flavonesapigenin, and apigenin-7-oglucoside. isovitexin, kaempferol3-O-beta-glucoside, luteolin-7-O-glucoside, and vitexin. It is furthercontemplated that in certain embodiments, a combination of bothmolecular, biochemical, and traditional methods can be used to identifyor monitor MSH1 suppression in plants.

IV. Recovery, Selfing, and Outcrossing of Progeny of MSH1 SuppressedPlants

A variety of methods that provide for suppression of MSH1 in a plantfollowed by recovery of progeny plants where MSH1 function is restoredare provided herein. In certain embodiments, such progeny plants can berecovered by downregulating expression of an MSH1-inhibiting transgeneor by removing the MSH1-inhibiting transgene with a transposase. Incertain embodiments of the methods provided herein, MSH1 is suppressedin a target plant or plant cell and progeny plants that express MSH1 arerecovered by traditional genetic techniques. In one exemplary andnon-limiting embodiment, progeny plants can be obtained by selfing aplant that is heterozygous for the transgene that provides for MSH1segregation. Selling of such heterozygous plants (or selfing ofheterozygous plants regenerated from plant cells) provides for thetransgene to segregate out of a subset of the progeny plant population.Where MSH1 is suppressed by use of a recessive mutation in an endogenousMSH1 gene (i.e. an msh1 plant), msh1/msh1 plants can, in yet anotherexemplary and non-limiting embodiment, be crossed to MSH1 plants andthen selfed to obtain progeny plants that are homozygous for afunctional, wild-type MSH1 allele. In other embodiments, MSH1 issuppressed in a target plant or plant cell and progeny plants thatexpress MSH1 are recovered by molecular genetic techniques. Non limitingand exemplary embodiments of such molecular genetic techniques include:i) downregulation of an MSH1 suppressing transgene under the control ofa regulated promoter by withdrawal of an inducer required for activityof that promoter or introduction of a repressor of that promoter; or,ii) exposure of the an MSH1 suppressing transgene flanked by transposaserecognition sites to the cognate transposase that provides for removalof that transgene.

In certain embodiments of the methods provided herein, progeny plantsderived from plants where MSH1 expression was suppressed that exhibitmale sterility, dwarfing, variegation, and/or delayed flowering time andexpress functional MSH1 are obtained and maintained as independentbreeding lines. It has been found that such phenotypes appear to sort,so that it is feasible to select a cytoplasmic male sterile plantdisplaying normal growth rate and no variegation, for example, or astunted, male fertile plant that is highly variegated. We refer to thisphenomenon herein as discrete variation (V_(D)). An exemplary andnon-limiting illustration of this phenomenon as it occurs in selfedplant populations that have lost an MSH1-inhibiting transgene bysegregation is provided in FIG. 6. It is further contemplated that suchindividual lines that exhibit discrete variation (V_(D)) can be obtainedby any of the aforementioned traditional genetic techniques, moleculargenetic techniques, or combinations thereof.

Individual lines obtained from plants where MSH1 expression wassuppressed that exhibit discrete variation (V_(D)) can be crossed toother plants to obtain progeny plants that lack the phenotypesassociated with discrete variation (V_(D)) (i.e. male sterility,dwarfing, variegation, and/or delayed flowering time). It hassurprisingly been found that progeny of such outcrosses can be selfed toobtain individual progeny lines that exhibit significant phenotypicvariation. Such phenotypic variation that is observed in theseindividual progeny lines derived from outcrosses of plants where MSH1expression was suppressed and that exhibit discrete variation to otherplants is herein referred to as “quantitative variation” (V_(Q)).Certain individual progeny plant lines obtained from the outcrosses ofplants where MSH1 expression was suppressed to other plants can exhibituseful phenotypic variation where one or more traits are improvedrelative to either parental line and can be selected. Useful phenotypicvariation that can be selected in such individual progeny linesincludes, but is not limited to, increases in fresh and dry weightbiomass relative to either parental line. An exemplary and non-limitingillustration of this phenomenon as it occurs in F2 progeny of outcrossesof plants that exhibit discrete variation to plants that do not exhibitdiscrete variation is provided in FIG. 6.

In certain embodiments, an outcross of an individual line exhibitingdiscrete variability can be to a plant that has not been subjected toMSH1 suppression but is otherwise isogenic to the individual lineexhibiting discrete variation. In certain exemplary embodiments, a lineexhibiting discrete variation is obtained by suppressing MSH1 in a givengermplasm and can outcrossed to a plant having that same germplasm thatwas not subjected to MSH1 suppression. In other embodiments, an outcrossof an individual line exhibiting discrete variability can be to a plantthat has not been subjected to MSH1 suppression but is not isogenic tothe individual line exhibiting discrete variation. Thus, in certainembodiments, an outcross of an individual line exhibiting discretevariability can also be to a plant that comprises one or morechromosomal polymorphisms that do not occur in the individual lineexhibiting discrete variability, to a plant derived from partially orwholly different germplasm, or to a plant of a different heterotic group(in instances where such distinct heterotic groups exist). It is alsorecognized that such an outcross can be made in either direction. Thus,an individual line exhibiting discrete variability can be used as eithera pollen donor or a pollen recipient to a plant that has not beensubjected to MSH1 suppression in such outcrosses. In certainembodiments, the progeny of the outcross are then selfed to establishindividual lines that can be separately screened to identify lines withimproved traits relative to parental lines. Such individual lines thatexhibit the improved traits are then selected and can be propagated byfurther selfing. An exemplary and non-limiting illustration of thisprocedure where F2 progeny of outcrosses of plants that exhibit discretevariation to plants that do not exhibit discrete variation are obtainedis provided in FIG. 6. Such F2 progeny lines are screened for desiredtrait improvements relative to the parental plants and lines exhibitingsuch improvements are selected.

V. Comparing and Selecting Altered Chromosomal Loci in Plants that canConfer a Useful Trait

Altered chromosomal loci that can confer useful traits can also beidentified and selected by performing appropriate comparative analysesof reference plants that do not exhibit the useful traits and testplants obtained from a parental plant or plant cell that had beensubjected to MSH1 suppression and obtaining either the altered loci orplants comprising the altered loci. It is anticipated that a variety ofreference plants and test plants can be used in such comparisons andselections. In certain embodiments, the reference plants that do notexhibit the useful trait include, but are not limited to, any of: a) awild-type plant; b) a distinct subpopulation of plants within a given F2population of plants of a given plant line (where the F2 population isany applicable plant type or variety obtained in the manner shown inFIG. 6); c) an F1 population exhibiting a wild type phenotype (where theF1 population is any applicable plant type or variety obtained in themanner shown in FIG. 6); and/or, d) a plant that is isogenic to theparent plants or parental cells of the test plants prior to suppressionof MSH1 in those parental plants or plant cells (i.e. the referenceplant is isogenic to the plants or plant cells that were later subjectedto MSH1 suppression to obtain the test plants). In certain embodiments,the test plants that exhibit the useful trait include, but are notlimited to, any of: a) any non-transgenic segregants that exhibit theuseful trait and that were derived from parental plants or plant cellsthat had been subjected to transgene mediated MSH1 suppression, b) adistinct subpopulation of plants within a given F2 population of plantsof a given plant line that exhibit the useful trait (where the F2population is any applicable plant type or variety obtained in themanner shown in FIG. 6); (c) any progeny plants obtained from the plantsof (a) or (b) that exhibit the useful trait; or d) a plant or plant cellthat had been subjected to MSH1 suppression that exhibit the usefultrait.

In general, an objective of these comparisons is to identify differencesin the small RNA profiles and/or methylation of certain chromosomal DNAloci between test plants that exhibit the useful traits and referenceplants that do not exhibit the useful traits. Altered loci thusidentified can then be isolated or selected in plants to obtain plantsexhibiting the useful traits.

In certain embodiments, altered chromosomal loci can be identified byidentifying small RNAs that are up or down regulated in the test plants(in comparison to reference plants). This method is based in part onidentification of altered chromosomal loci where small interfering RNAsdirect the methylation of specific gene targets by RNA-directed DNAmethylation (RdDM). The RNA-directed DNA methylation (RdDM) process hasbeen described (Chinnusamy V et al. Sci China Ser C-Life Sci. (2009)52(4): 331-343). Any applicable technology platform can be used tocompare small RNAs in the test and reference plants, including, but notlimited to, microarray-based methods (Franco-Zorilla et al. Plant J.2009 59(5):840-50), deep sequencing based methods (Wang et al. The PlantCell 21:1053-1069 (2009)), and the like.

In certain embodiments, altered chromosomal loci can be identified byidentifying histone proteins associated with a locus and that aremethylated or acylated in the test plants (in comparison to referenceplants). The analysis of chromosomal loci associated with methylated oracylated histones can be accomplished by enriching and sequencing thoseloci using antibodies that recognize methylated or acylated histones.Identification of chromosomal regions associated with methylation oracetylation of specific lysine residues of histone H3 by usingantibodies specific for H3K4me3, H3K9ac, H3K27me3, and H3K36me3 has beendescribed (Li et al., Plant Cell 20:259-276, 2008; Wang et al. The PlantCell 21:1053-1069 (2009).

In certain embodiments, altered chromosomal loci can be identified byidentifying chromosomal regions (genomic DNA) that has an alteredmethylation status in the test plants (in comparison to referenceplants). An altered methylation status can comprise either the presenceor absence of methylation in one or more chromosomal loci of a testplant comparison to a reference plant. Any applicable technologyplatform can be used to compare the methylation status of chromosomalloci in the test and reference plants. Applicable technologies foridentifying chromosomal loci with changes in their methylation statusinclude, but not limited to, methods based on immunoprecipitation of DNAwith antibodies that recognize 5-methylcytidine, methods based on use ofmethylation dependent restriction endonucleases and PCR such asMcrBC-PCR methods (Rabinowicz, et al. Genome Res. 13: 2658-2664 2003; Liet al., Plant Cell 20:259-276, 2008), sequencing of bisulfite-convertedDNA (Frommer et al. Proc. Natl. Acad. Sci. U.S.A. 89 (5): 1827-31; Tostet al. BioTechniques 35 (1): 152-156, 2003), methylation-specific PCRanalysis of bisulfite treated DNA (Herman et al. Proc. Natl. Acad. Sci.U.S.A. 93 (18): 9821-6, 1996), deep sequencing based methods (Wang etal. The Plant Cell 21:1053-1069 (2009)), methylation sensitive singlenucleotide primer extension (MsSnuPE; Gonzalgo and Jones Nucleic AcidsRes. 25 (12): 2529-2531, 1997), fluorescence correlation spectroscopy(Umezu et al. Anal Biochem. 415(2):145-50, 2011), single molecule realtime sequencing methods (Flusberg et al. Nature Methods 7, 461-465),high resolution melting analysis (Wojdacz and Dobrovic (2007) NucleicAcids Res. 35 (6): e41), and the like.

VI. Introducing a Chromosomal Modification Associated with a UsefulTrait into a Plant

Methods for introducing various chromosomal modifications that canconfer a useful trait into a plant, as well as the plants, plant parts,and products of those plant parts are also provided herein. Chromosomalalterations and/or chromosomal mutations induced by suppression of MSH1can be identified as described herein. Once identified, chromosomalmodifications including, but not limited to, chromosomal alterations,chromosomal mutations, or transgenes that provide for the same geneticeffect as the chromosomal alterations and/or chromosomal mutationsinduced by suppression of MSH1 can be introduced into host plants toobtain plants that exhibit the desired trait. In this context, the “samegenetic effect” means that the introduced chromosomal modificationprovides for an increase and/or a reduction in expression of one or moreendogenous plant genes that is similar to that observed in a plant thathas been subjected to MSH1 suppression and exhibits the useful trait. Incertain embodiments where an endogenous gene is methylated in a plantsubjected to MSH1 suppression and exhibits both reduced expression ofthat gene and a useful trait, chromosomal modifications in other plantsthat also result in reduced expression of that gene and the useful traitare provided. In certain embodiments where an endogenous gene isdemethylated in a plant subjected to MSH1 suppression and exhibits bothincreased expression of that gene and a useful trait, chromosomalmodifications in other plants that also result in increased expressionof that gene and that useful trait are provided.

In certain embodiments, the chromosomal modification that is introducedis a chromosomal alteration. Chromosomal alterations including, but notlimited to, a difference in a methylation state can be introduced bycrossing a plant comprising the chromosomal alteration to a plant thatlacks the chromosomal alteration and selecting for the presence of thealteration in F1, F2, or any subsequent generation progeny plants of thecross. In still other embodiments, the chromosomal alterations inspecific target genes can be introduced by expression of a siRNA orhairpin RNA targeted to that gene by RNA directed DNA methylation(Chinnusamy V et al. Sci China Ser C-Life Sci. (2009) 52(4): 331-343;Cigan et al. Plant J 43 929-940, 2005; Heilersig et al. (2006) Mol GenetGenomics 275 437-449; Miki and Shimamoto, Plant Journal 56(4):539-49;Okano et al. Plant Journal 53(1):65-77, 2008).

In certain embodiments, the chromosomal modification is a chromosomalmutation. Chromosomal mutations that provide for reductions or increasesin expression of an endogenous gene of a chromosomal locus can include,but are not limited to, insertions, deletions, and/or substitutions ofnucleotide sequences in a gene. Chromosomal mutations can result indecreased expression of a gene by a variety of mechanisms that include,but are not limited to, introduction of missense codons, frame-shiftmutations, premature translational stop codons, promoter deletions,mutations that disrupt mRNA processing, and the like. Chromosomalmutations that result in increased expression of a gene include, but arenot limited to, promoter substitutions, removal of negative regulatoryelements from the gene, and the like. Chromosomal mutations can beintroduced into specific loci of a plant by any applicable method.Applicable methods for introducing chromosomal mutations in endogenousplant chromosomal loci include, but are not limited to, homologousdouble stranded break repair (Wright et al., Plant J. 44, 693, 2005;D'Halluin, et al., Plant Biotech. J. 6:93, 2008), non-homologous endjoining or a combination of non-homologous end joining and homologousrecombination (reviewed in Puchta, J. Exp. Bot. 56, 1, 2005; Wright etal., Plant J. 44, 693, 2005), meganuclease-induced, site specific doublestranded break repair (WO/06097853A1, WO/06097784A1, WO/04067736A2, U.S.20070117128A1), and zinc finger nuclease mediated homologousrecombination (WO 03/080809, WO 05/014791, WO 07014275, WO 08/021207).In still other embodiments, desired mutations in endogenous plantchromosomal loci can be identified through use of the TILLING technology(Targeting Induced Local Lesions in Genomes) as described (Henikoff etal., Plant Physiol. 2004, 135:630-636).

In other embodiments, chromosomal modifications that provide for thedesired genetic effect can comprise a transgene. Transgenes that canresult in decreased expression of an gene by a variety of mechanismsthat include, but are not limited to, dominant-negative mutants, a smallinhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA,and/or an anti-sense RNA and the like. US patents incorporated herein byreference in their entireties that describe suppression of endogenousplant genes by transgenes include U.S. Pat. No. 7,109,393, U.S. Pat. No.5,231,020 and U.S. Pat. No. 5,283,184 (co-suppression methods); and U.S.Pat. No. 5,107,065 and U.S. Pat. No. 5,759,829 (antisense methods). Incertain embodiments, transgenes specifically designed to producedouble-stranded RNA (dsRNA) molecules with homology to the endogenousgene of a chromosomal locus can be used to decrease expression of thatendogenous gene. In such embodiments, the sense strand sequences of thedsRNA can be separated from the antisense sequences by a spacersequence, preferably one that promotes the formation of a dsRNA(double-stranded RNA) molecule. Examples of such spacer sequencesinclude, but are not limited to, those set forth in Wesley et al., PlantJ., 27(6):581-90 (2001), and Hamilton et al., Plant J., 15:737-746(1998). Vectors for inhibiting endogenous plant genes withtransgene-mediated expression of hairpin RNAs are disclosed in U.S.Patent Application Nos. 20050164394, 20050160490, and 20040231016, eachof which is incorporated herein by reference in their entirety.

Transgenes that result in increased expression of a gene of achromosomal locus include, but are not limited to, a recombinant genefused to heterologous promoters that are stronger than the nativepromoter, a recombinant gene comprising elements such as heterologousintrons, 5′ untranslated regions, 3′ untranslated regions that providefor increased expression, and combinations thereof. Such promoter,intron, 5′ untranslated, 3′ untranslated regions, and any necessarypolyadenylation regions can be operably linked to the DNA of interest inrecombinant DNA molecules that comprise parts of transgenes useful formaking chromosomal modifications as provided herein.

Exemplary promoters useful for expression of transgenes include, but arenot limited to, enhanced or duplicate versions of the viral CaMV35S andFMV35S promoters (U.S. Pat. No. 5,378,619, incorporated herein byreference in its entirety), the cauliflower mosaic virus (CaMV) 19Spromoters, the rice Act1 promoter and the Figwort Mosaic Virus (FMV) 35Spromoter (U.S. Pat. No. 5,463,175; incorporated herein by reference inits entirety). Exemplary introns useful for transgene expressioninclude, but are not limited to, the maize hsp70 intron (U.S. Pat. No.5,424,412; incorporated by reference herein in its entirety), the riceAct1 intron (McElroy et al., 1990, The Plant Cell, Vol. 2, 163-171), theCAT-1 intron (Cazzonnelli and Velten, Plant Molecular Biology Reporter21: 271-280, September 2003), the pKANNIBAL intron (Wesley et al., PlantJ. 2001 27(6):581-90; Collier et al., 2005, Plant J 43: 449-457), thePIV2 intron (Mankin et al. (1997) Plant Mol. Biol. Rep. 15(2): 186-196)and the “Super Ubiquitin” intron (U.S. Pat. No. 6,596,925, incorporatedherein by reference in its entirety; Collier et al., 2005, Plant J 43:449-457). Exemplary polyadenylation sequences include, but are notlimited to, and Agrobacterium tumor-inducing (Ti) plasmid nopalinesynthase (NOS) gene and the pea ssRUBISCO E9 gene polyadenylationsequences.

VII. Screening and Selection of Outcrossed Progeny of MSH1 SuppressedPlants or Plants comprising Modified Chromosomal Loci that ExhibitImproved or Useful Traits

Plant lines obtained by the methods provided herein can be screened andselected for a variety of useful traits by using a wide variety oftechniques. In particular embodiments provided herein, individualprogeny plant lines obtained from the outcrosses of plants where MSH1expression was suppressed to other plants are screened and selected forthe desired useful traits.

In certain embodiments, the screened and selected trait is improvedplant yield. In certain embodiments, such yield improvements areimprovements in the yield of a plant line relative to one or moreparental line(s) under non-stress conditions. Non-stress conditionscomprise conditions where water, temperature, nutrients, minerals, andlight fall within typical ranges for cultivation of the plant species.Such typical ranges for cultivation comprise amounts or values of water,temperature, nutrients, minerals, and/or light that are neitherinsufficient nor excessive. In certain embodiments, such yieldimprovements are improvements in the yield of a plant line relative toparental line(s) under abiotic stress conditions. Such abiotic stressconditions include, but are not limited to, conditions where water,temperature, nutrients, minerals, and/or light that are eitherinsufficient or excessive. Abiotic stress conditions would thus include,but are not limited to, drought stress, osmotic stress, nitrogen stress,phosphorous stress, mineral stress, heat stress, cold stress, and/orlight stress. In this context, mineral stress includes, but is notlimited to, stress due to insufficient or excessive potassium, calcium,magnesium, iron, manganese, copper, zinc, boron, aluminum, or silicon.In this context, mineral stress includes, but is not limited to, stressdue to excessive amounts of heavy metals including, but not limited to,cadmium, copper, nickel, zinc, lead, and chromium.

Improvements in yield in plant lines obtained by the methods providedherein can be identified by direct measurements of wet or dry biomassincluding, but not limited to, grain, lint, leaves, stems, or seed.Improvements in yield can also be assessed by measuring yield relatedtraits that include, but are not limited to, 100 seed weight, a harvestindex, and seed weight. In certain embodiments, such yield improvementsare improvements in the yield of a plant line relative to one or moreparental line(s) and can be readily determined by growing plant linesobtained by the methods provided herein in parallel with the parentalplants. In certain embodiments, field trials to determine differences inyield whereby plots of test and control plants are replicated,randomized, and controlled for variation can be employed (Giesbrecht F Gand Gumpertz M L. 2004. Planning, Construction, and Statistical Analysisof Comparative Experiments. Wiley. New York; Mead, R. 1997. Design ofplant breeding trials. In Statistical Methods for Plant VarietyEvaluation. eds. Kempton and Fox. Chapman and Hall. London.). Methodsfor spacing of the test plants (i.e. plants obtained with the methods ofthis invention) with check plants (parental or other controls) to obtainyield data suitable for comparisons are provided in references thatinclude, but are not limited to, any of Cullis, B. et al. J. Agric.Biol. Env. Stat. 11:381-393; and Besag, J. and Kempton, R A. 1986.Biometrics 42: 231-251.).

In certain embodiments, the screened and selected trait is improvedresistance to biotic plant stress relative to the parental lines. Bioticplant stress includes, but is not limited to, stress imposed by plantfungal pathogens, plant bacterial pathogens, plant viral pathogens,insects, nematodes, and herbivores. In certain embodiments, screeningand selection of plant lines that exhibit resistance to fungal pathogensincluding, but not limited to, an Alternaria sp., an Ascochyta sp., aBotrytis sp.; a Cercospora sp., a Colletotrichum sp., a Diaporthe sp., aDiplodia sp., an Erysiphe sp., a Fusarium sp., Gaeumanomyces sp.,Helminthosporium sp., Macrophomina sp., a Nectria sp., a Peronosporasp., a Phakopsora sp., Phialophora sp., a Phoma sp., a Phymatotrichumsp., a Phytophthora sp., a Plasmopara sp., a Puccinia sp., a Podosphaerasp., a Pyrenophora sp., a Pyricularia sp, a Pythium sp., a Rhizoctoniasp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., a Thielaviopsissp., an Uncinula sp, a Venturia sp., and a Verticillium sp. is provided.In certain embodiments, screening and selection of plant lines thatexhibit resistance to bacterial pathogens including, but not limited to,an Erwinia sp., a Pseudomonas sp., and a Xanthamonas sp. is provided. Incertain embodiments, screening and selection of plant lines that exhibitresistance to insects including, but not limited to, aphids and otherpiercing/sucking insects such as Lygus sp., lepidoteran insects such asArmigera sp., Helicoverpa sp., Heliothis sp., and Pseudoplusia sp., andcoleopteran insects such as Diabroticus sp. is provided. In certainembodiments, screening and selection of plant lines that exhibitresistance to nematodes including, but not limited to, Meloidogyne sp.,Heterodera sp., Belonolaimus sp., Ditylenchus sp., Globodera sp.,Naccobbus sp., and Xiphinema sp. is provided.

Other useful traits that can be obtained by the methods provided hereininclude various seed quality traits including, but not limited to,improvements in either the compositions or amounts of oil, protein, orstarch in the seed. Still other useful traits that can be obtained bymethods provided herein include, but are not limited to, increasedbiomass, non-flowering, male sterility, digestability, seed fillingperiod, maturity (either earlier or later as desired), reduced lodging,and plant height (either increased or decreased as desired).

In addition to any of the aforementioned traits, particularly usefultraits for sorghum that can be obtained by the methods provided hereinalso include, but are not limited to: i) agronomic traits (floweringtime, days to flower, days to flower-post rainy, days to flower-rainy;ii) fungal disease resistance (sorghum downy mildewresistance—glasshouse, sorghum downy mildew resistance-field, sorghumgrain mold, sorghum leaf blight resistance, sorghum rust resistance;iii) grain related trait: (Grain dry weight, grain number, grain numberper square meter, Grain weight over panicle. seed color, seed luster,seed size); iv) growth and development stage related traits (basaltillers number, days to harvest, days to maturity, nodal tillering,plant height, plant height-postrainy); v) infloresence anatomy andmorphology trait (threshability); vi) Insect damage resistance (sorghumshoot fly resistance-post-rainy, sorghum shoot fly resistance-rainy,sorghum stem borer resistance); vii) leaf related traits (leaf color,leaf midrib color, leaf vein color, flag leaf weight, leaf weight, restof leaves weight); viii) mineral and ion content related traits (shootpotassium content, shoot sodium content); ix) panicle related traits(number of panicles, panicle compactness and shape, panicle exertion,panicle harvest index, panicle length, panicle weight, panicle weightwithout grain, panicle width); x) phytochemical compound content (plantpigmentation); xii) spikelet anatomy and morphology traits (glume color,glume covering); xiii) stem related trait (stem over leaf weight, stemweight); and xiv) miscellaneous traits (stover related traits,metabolised energy, nitrogen digestibility, organic matterdigestibility, stover dry weight).

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Construction of Transgenic Plants that provide for Suppressionof MSH1

A vector that provides for suppression of MSH1 in tomato and tobacco wasconstructed as follows. A segment encoding amino acids 651-870 of theMSH1 protein was derived from a tomato EST sequence (SEQ ID NO:5) byusing the primer sequences TOM-CD1F (5′-CGCAGGTATCACGAGGCAAGTGCTAAGG-3;SEQ ID NO:11) and TOM-CD1R(5′-ATCCCCAAACAGCCAATTTCGTCCAGGATCCCCAAACAGCCAATTTCGTCCAGG-3; SEQ IDNO:12) and cloned in forward and reverse orientation, separated by anintron sequence. The base vector, pUCRNAi-intron harbors the secondintron of the Arabidopsis small nuclear riboprotein (At4g02840; SEQ IDNO: 13). The CaMV35S promoter and transcription terminator regulateexpression of the construction and the neomycin phosphotransferase II(nptII) reporter gene, and the insert is flanked by right border andleft border integration sequences. Agrobacterium tumefaciens strainC58C1/pMP90 (28) was used for transformation in tobacco (Horsch R B, etal. (1985) Science 227:1229-1231) and tomato (McCormick et al. 1986)Plant Cell Rep 5:81-84).

Millet and sorghum RNAi lines were derived by similar procedures andmaterials, with transformations and plant regeneration carried outaccording to the procedures of Howe et al. (Plant Cell Rep 25:784-91,2006). The RNAi vector for millet was directed against the millet MSH1gene whereas the RNAi vector for sorghum was directed against thesorghum MSH1 gene (SEQ ID NO: 6). Segments encoding 157 amino acids fromthe MSH1 C-terminal were amplified from total cDNA of pearl millet andsorghum using primers: zm-msf8 (5′-GGTTGAGGAGCCTGAATCTCTGAAGAAC-3′; SEQID NO:15) and zm-msr8 (5′-CTCGCCAGAGATTCGAGATATACCGAAG-3′; SEQ IDNO:16). PCR products were cloned in forward and reverse orientation,separated by an intron sequence. The base vector, pUCRNAi-intron, whichharbors the second intron of the Arabidopsis small nuclear riboprotein(At4g02840; SEQ ID NO: 13), was provided by H. Cerutti (University ofNebraska, Lincoln, Nebr.). The vector pPTN290, a derivative of pPZP212(Hajdukiewicz et al. 1994, Plant Mol Biol.; 25(6):989-94), was used tointroduce the Msh1-RNAi cassettes under the control of the maizeubiquitin 1 promoter coupled with its first intron, and itstranscription is terminated by CaMV 35S terminator. The CaMV 35Spromoter and terminator regulate the expression of the neomycinphosphotransferase II (nptII) reporter gene, and the insert is flankedby right border and left border integration sequences. The Agrobacteriumtumefaciens strain NTL4 (Luo Z-Q et al., 2001, Mol Plant MicrobeInteract., 14(1):98-103) was used for inoculating embryos from pearlmillet maintainer Tift23 DBE1 and sorghum Tx430 lines. Detailedtransformation procedures used for pearl millet are the same as forsorghum (Howe et al., 2006, Plant Cell Rep 25:784-91).

Example 2 Phenotypic Effects of MSH1 Suppression

MSH1 expression suppressed transgenically by use of RNAi in five plantspecies: soybean (Glycine max (L.) Merr), tomato (Solanum lycopersicumL), tobacco (Nicotiana tabacum L.), millet (Pennisetum glaucum (L.) R.Br.) and sorghum (Sorghum bicolor (L.) Moench). In each case, similarchanges were observed, including cytoplasmic male sterility, evidence ofvariegation and altered chloroplast development, reduced growth rate anddwarfing, altered flowering time or non-flowering, enhanced branching,reduced flavonoid biosynthesis and lack of anthocyanins, enhancedpathogen susceptibility, and altered leaf morphologies (see FIG. 1).Variegation, dwarfing, and mitochondrial DNA rearrangements are alsoobserved in various plants subjected to MSH1 suppression as shown inFIGS. 2, 3, and 4, respectively. Physiologically, plants show reducedATP and enhanced ROS levels, reduced mitochondrial motility, enhancedmitophagy, expression of stress response pathways, and altered cytokininand GA metabolism (ROS data in FIG. 5).

The striking phenotypic similarities among plant species indicate thatmany of the msh1-associated changes are programmed responses. Transcriptand metabolic analyses have identified several pathways associated withthe emerging phenotypes (Table 1). Sorghum and Arabidopsis transcriptprofiling experiments show reduced expression of cell cycle genes,altered flowering gene expression (FLC), and enhanced GA catabolism(GA20-ox2 and GA20-ox6) in the reduced growth phenotypes. Plants arerestored in growth rate and flower induction with the application ofgibberellic acid.

TABLE 1 Sample transcript/metabolic profiling results in Arabidopsisshowing correspondence in pathway changes. Transcript ProfilingMetabolic Profiling AGI Gene msh1* metabolite Col-0 msh1 A.Redox/oxidative stress response AT3G22370 AOX1A 2.2 Glutathione† 22,52033,322 AT5G20230 ATBCB 10.9 Ascorbate† 289,996 460,261 AT2G21640 OxidStress Response 2.9 phosphate 12.3M 32.1M AT4G20830 FAD-binding domainprotein 2.6 B. Photosynthesis genes

PSBO-1 −1.3

26,969.4 N.D.

PSBO-2 −1.4

49,427.8 N.D.

PSAD-1 −1.6

PSBP-2 −2 C. GA response AT1G30040 ATGA20X2 (GA catabolism) 1.7

11 ng/g DW N.D. AT1G02400 ATGA20X6 (GA catabolism) 9.3

 7 ng/g DW N.D.

GA-regulated protein −3.3 *Fold change of levels in msh1 relative toCol-0, †values are normalized raw area count from mass spectrometeranalysis, N.D. non detectable A limited dataset is shown. Shadingindicates downregulation in msh1.

Example 3 Genetic Analysis of Tx430 Sorghum Lines following Exposure toand Loss of the MSH1 RNAi Transgene by Segregation

A non-transgenic, highly dwarfed, delayed flowering and variegated TX430sorghum plant was obtained from a segregation population of progenyplants from a parental Tx430 sorghum plant that was heterozygous for atransgene that inhibits MSH1 expression by RNA interference (RNAi).Tx430 was the original genotype used to obtain the transgenic sorghumplant comprising the transgene that inhibits MSH1 expression. Crossingof this non-transgenic, highly dwarfed, delayed flowering and variegatedTX430 sorghum plant by isogenic TX430 wildtype as pollen parent,produced a wildtype F1 phenotype that showed no evidence of the originaldwarfing, delay in flowering or variegation phenotypes (FIG. 6). Thiswas a surprising result, since we had assumed the RNAi-induced changesto be organellar, and anticipated maternal transmission of thephenotypes. Introduction of the wildtype genome neutralized the originalRNAi-induced effects. The F2 population, derived by self-pollination ofthese F1 plants, produced a broad distribution of phenotypic variation,referred to as quantitative variation (VQ), some of which is describedin Table 2. SAS PROC MIXED was used for all analyses in Table 2. Eachtrait was analyzed with the fixed effect of line in the model andheterogeneous variances among the lines were assumed and estimated,along with standard errors of the estimates. A chi-square test of theheterogeneous variance model against the homogeneous variance model wasperformed. A significant chi-square value indicates statisticallysignificant differences among line variances. While a small proportion(ca. 1/50 plants) shows the dwarfed, variegated phenotype, and about 50%show cytoplasmic male sterility as a likely mitochondrial genetic lesion(Hanson and Bentolila, 2004), a large proportion of the population showssignificant quantitative variation in aboveground fresh and dry weightbiomass, panicle weight, and other useful agronomic features.Particularly intriguing in these data is the observed capacity withinthe population to out-perform either parent for several traits. Therange of diversity cannot reasonably be accounted for by nuclear geneticvariation, since the original cross is TX430×TX430 (made in thegreenhouse with bagged panicles).

TABLE 2 Assessment of phenotypic variation in Sorghum Chi- Line N¹Lsmean Variance SE variance Square² P-value Plant F1 31 156.65  1195.5308.68 156.98 <0.0001 Height (cm) Plant F2 274 143.63  1400.33 119.86Height (cm) Plant Dwarf 55 48.29   61.17 11.77 Height (cm) PlantWildtype 18 131.11   32.58 11.17 Height (cm) Panicle F1 13 27.154  11.81 4.82 4.75 0.0931 Length (cm) Panicle F2 275 27.171   17.20 1.47Length (cm) Panicle Wild 11 26.636   5.85 2.61 Length Type (cm) PanicleF1 16 46.63  252.65 92.25 14.49 0.0007 Weight (grams) Panicle F2 36845.26  365.78 27.00 Weight (grams) Panicle Wild 17 33.53   67.51 23.87Weight Type (grams) Dry F1 3 294.7 12258³ 12258 16.46 0.0009 Biomass(grams) Dry F2 52 224.8  3023.4 598.7 Biomass (grams) Dry Dwarf 11 195.8 2696.6 1205.9 Biomass (grams) Dry Wild 10 193.6  283.1 133.5 BiomassType (grams) ¹N = number of observations in a line ²Chi-square test istest for differences among line variances ³The unusually high varianceis the consequence of small sample size for this trait.

Example 4 Analysis of Arabidopsis MSH1/MSH1 F3 Progeny of amsh1/msh1×MSH1/MSH1 Cross

In these experiments, the recessive msh1 mutation was removed bysegregation. The recessive msh1/msh1 Columbia ecotype parent was firstcrossed to wild type Columbia ecotype plants as pollen donor (Col-0msh1×Col-0wt) to obtain an F1 population of msh1/MSH1 plants. The F1progeny were (selfed to obtain an F2 population segregating for themsh1locus. MSH1/MSH1 F2 progeny were selected from the F2 population andselfed to obtain MSH1/MSH1 F3 progeny of the selected MSH1/MSH1 F2parent.

To assess phenotypic variation in the selected F3 MSH1/MSH1 Arabidopsislines, measurements were averaged from four plants each of wildtypeCol-0 and the selected F3 progeny line as shown in Table 3. Freshbiomass was total aboveground leaf tissue, base diameter was thediameter of root-stem transition zone, and stalk diameter was thediameter of the floral stalk. Each parameter showed a 20-24% increase inthe selected F3 progeny line, even though the two plant populations(i.e. Col-O and MSH1/MSH1 F3) progeny should be genetically identical.Plants from each group were selected to represent the same stage ofdevelopment and same number of leaves (average of 48 leaves per plant ineach group). The data of Table 3 and plants shown in FIG. 7 representone selected F3 population. Other selected F3 populations (not shown)demonstrated uniformly lower average growth relative to wildtype.

One MSH1/MSH1 F3 progeny derived from the Col-0 msh1×Col-0wt crossshowed markedly enhanced growth as shown in FIG. 7 and Table 3. Suchmarkedly enhanced growth resembles hybrid vigor in that the F3 progenyof the cross exhibit increased growth relative to the Col-O parentalgermplasm. However, these experiments can be distinguished frominstances where hybrid vigor is obtained by crossing parental lines oftwo distinct heterotic genetic backgrounds since the two parental linesused here both had Columbia ecotype genetic backgrounds and differedonly in the presence of the recessive msh1 mutation in one of theColumbia ecotype parents.

TABLE 3 Assessment of phenotypic variation in Arabidopsis. Col-0 msh1 ×Col-0 F₃ (wild-type parent) (MSH1 positive progeny) Fresh biomass (g)4.9 6.3 Base diameter (mm) 2.2 2.9 Stalk diameter (mm) 1.6 2.0

Example 5 Variation in Plant Height, Panicle Weight, and Grain Yield inIndividual Sorghum Plants in an F2 Population Obtained from an Outcrossto MSH1—Suppressed Sorghum

F2 populations of sorghum plants derived from parental Tx430 sorghumplants that had been subjected to MSH1 suppression as describe in FIG. 6and Example 3 were assayed for variation in plant height (FIG. 8),panicle weight (FIG. 9), and grain yield (FIG. 10) by comparing thevalues for individual plants in the population.

Significant variation was observed between individual plants within theF2 population. More specifically, certain sorghum lines exhibiteddistinctive bi-phasic distributions of plants within the F2 populationswith respect to these traits. For example, the F2 population of sorghumline GAII-11 exhibited one subpopulation of plants with plant heightbetween about 105 and 125 cM and another sub-population of plants with aplant height between about 185 to 215 cM. These subpopulations wererepresented by “peaks” in the FIG. 8 plot. Similar distributions ofsubpopulations are also observed for sorghum lines GA11-15, GA11-28 andGA11-24 in the FIG. 8 plot. For the GA11-11, GA11-15, GA11-28 andGA11-24 F2 populations, one set of sub-populations either overlapped orhad a value less than that of the wild-type TA430 control plant heightswhile another sub-population had a value that was clearly greater thanthat of the wild-type PA430 control plants (FIG. 8). Subpopulationsand/or individual plants in the GA11-11, GA11-15, GA11-28 and GA11-24 F2populations also exhibited panicle weights and grain yields that eitheroverlapped or had a value less than that of the wild-type TA430 controlplant heights while other sub-populations or plants had a value that wasclearly greater than that of the wild-type PA430 control plants (FIGS. 9and 10).

It is concluded that differences in sorghum plant height, panicleweight, and grain yield are observed between: a) distinct subpopulationsof plants within a given F2 population of sorghum plants of a givensorghum line; and/or: b) a distinct sub-populations of plants within agiven F2 population of sorghum plants of a given sorghum line and thewild-type parental control line. It is further contemplated that thosesub-populations of sorghum plants that exhibit desirable increases inplant height, panicle number, and/or grain yield may comprise certaindifferences in their chromosomal DNA methylation state, theirchromosomal DNA sequence, post-translation modifications of a histoneprotein associated with a chromosomal locus, or any combination thereofthat either contribute directly to such useful traits (i.e. have adirect causal relationship to the useful trait) or are associated byeither genetic or epigenetic linkage(s) to loci that contribute directlyto such desirable traits.

Example 6 Characterization of Small RNA Profiles and DNA MethylationState in Plants Exhibiting Useful Traits Associated with MSH1Suppression

A comparison of small RNA profiles and DNA methylation states inreference plants that do not exhibit a useful phenotype and test plantscomprising an altered chromosomal locus associated with a useful traitcan be used to identify altered chromosomal loci. Methods for makingsuch comparisons that can be generalized to a variety of plants areprovided in this example.

In a particular exemplary embodiment, the small RNA profiles and DNAmethylation states of various chromosomal loci in: a) distinctsubpopulations of plants within a given F2 population of sorghum plantsof a given sorghum line; and/or: b) a distinct sub-populations of plantswithin a given F2 population of sorghum plants of a given sorghum lineand the wild-type parental control line; are compared. The objective ofthese comparisons is to identify differences in the small RNA profilesand/or methylation of certain chromosomal DNA loci between those sorghumplants that exhibit the useful traits and sorghum plants that do notexhibit the useful traits. Such differences can then be used to identifysRNAs or chromosomal loci that either contribute directly to such usefultraits or are associated by either genetic linkage(s) or through anepigenetic mechanism to loci that contribute directly to such usefultraits. Sorghum plants that will be examined can include wild typeplants, plants from distinct sub-populations and/or individual plants inthe GA11-11, GA11-15, GA11-28 and GA11-24 or other sorghum line F2populations that exhibit plant heights, panicle weights, and/or grainyields that either overlap or have a value less than that of thewildtype TA430 control plant heights as well as plants from distinctsub-populations and/or individual plants in the GA11-11, GA11-15,GA11-28 and GA11-24 or other sorghum line F2 populations that exhibitplant heights, panicle weights, and/or grain yields that are clearlygreater than that of the wild-type TA430 control plants. Such plants andsuch sub-populations are exemplarily described in the preceding Example5 and in FIGS. 8, 9, and 10.

The small RNA (sRNA) profiles of wild type sorghum (Tx430), F1 sorghum,and selected F2 sorghum plants derived from different sub-populationsare determined. Sorghum sub-populations or plants that will be examinedcan include wild type plants, and subpopulations and/or individualplants in the GA11-11, GA11-15, GA11-28 and GA11-24 or other sorghum F2populations as described above. For example, certain sorghum populationssubjected to MSH1 suppression can exhibit panicle weights and grainyields that either overlap or have a value less than that of thewild-type TA430 control plant heights while other sorghumsub-populations or plants can a value that was clearly greater than thatof the wild-type TA430 control plants as shown in FIGS. 9 and 10 can besubjected to deep sequencing to identify the types (qualitativeanalysis) and relative amounts (quantitative analysis) of sRNAs presentin these various plant lines. Such qualitative and quantitative analysescan then be used to establish correlations between the presence orabsence of a given phenotype and the presence, absence, or relativeabundance of a given sRNA.

Deep sequencing techniques to characterize sRNA populations can bedetermined as described by methods including but not limited to thosedescribed by Zhou et al. PLoS One. 2010; 5(12): e15224; or Glazov et al.PLoS One. 2009 Jul. 27; 4(7):e6349. In certain embodiments, threebiological replicates can be sequenced for each sample and sRNAlibraries can be prepared and sequenced according to an Illumina™protocol. Briefly, low-molecular weight sRNAs (17-27 nt in length) canbe isolated from total RNA by size fractionation. Following ligation of3′ and 5′ adaptors to sRNAs, RT-PCR will be performed to construct thesRNA library. The library will be purified and validated according tothe Illumina™ protocol and Illumina™-based deep sequencing of thelibrary can be performed

Following removal of common sequences (rRNA, tRNA, snRNA, and snoRNA),the remaining sRNA sequences will be subjected to several analyses. Thefirst analysis is to assess distribution of sRNAs in the genome, withthe expectation of identifying altered sRNA distribution by disruptionof MSH1 function. Analysis of genomic clustering will be used to examinethe distribution of sRNA-generating loci in the genome. An sRNA clusterwill be defined as a group of sRNAs, in which each small RNA is <100 ntfrom its nearest neighbor as described in Johnson et al. (2009). Basedon this definition, sRNAs at the ends of a cluster are >100 nt away fromthe next nearest small RNA outside the cluster (Johnson et al., 2009).The differential expression of siRNA signatures among different plantlines can be compared to gain insight into their relationship withdisrupted MSH1 function. This will be accomplished by comparing therelative abundance of miRNAs or siRNAs in each library derived from eachplant line. The SAMseq method can be used to perform statisticalanalysis of significant levels of differential expression. Several sRNAsthat exhibit differential expression patterns in deep-sequencinganalysis can be selected for validation using RNA gel blot analysis.

To gain information on the relationship between alterations in DNAmethylation and sRNAs levels in various samples, regions containing DNAmethylation (described below) can be mapped against the sRNAs obtainedfrom this study and other publicly available databases, to identifyregions containing DNA methylation that are potentially targeted bysRNA.

The sRNA and DNA methylation profiles obtained from different lines canbe compared to determine whether alterations in DNA methylation contentcorrelate with changes in sRNA abundance in various plant samples thatexhibit different MSH-1 induced phenotypes. One concern in such analysesis that sRNAs may be too short to be detected. sRNAs are typicallygenerated from much longer transcripts in plants. Therefore, one canexpand analyses of DNA methylation to 500 by on either side of thechromosomal locus containing sRNAs as reported (Wang et al., 2009). Thisanalysis would indicate whether DNA methylation could potentially beinduced by sRNAs. Such studies can be used to identify detectablealterations in the sRNA population that alter genome methylationpatterning that can result from MSH1 suppression. Any of the sRNAsand/or genomic regions identified in such studies can then be suppressedand/or up-regulated using transgenic or other genomic alteration-basedapproaches to obtain desirable phenotypes that can result from MSH1suppression.

Association of useful phenotypes induced by MSH1 suppression in variousplants and plant lines with chromosomal alterations can also bedetermined by methyl C detection in whole genome bisulfite sequencingexperiments. The genomic bisulfite deep sequencing method (Lister 2009)can be used to obtain a whole-genome view of all possible methylatedcytosines in the genomes of plants subjected to MSH1 suppressionincluding, but not limited to, those plants exhibiting desirablephenotypes or undesirable phenotypes, and suitable control plantsincluding, but not limited to, parental lines that have not beensubjected to MSH1 suppression. In an exemplary method, about fivemicrograms of genomic DNA can be isolated and spiked with 25 nanogramsof unmethylated lambda DNA that serves as an internal control for theefficiency of bisulfite conversion of non-methylated cytosinenucleotides to uracils. The DNA can be sonicated to an average length ofabout 300 by and a DNA library can be constructed. An exemplary methodthat follows an Illumina™ Paired End protocol comprising modificationswhere the end repair cocktail do not contain dCTP and the adapterscontain methylated cytosines (Illumina™) can be used. Bisulfiteconversion of the adapter-ligated DNA can be followed by limited cyclesof PCR with a uracil insensitive PfuTurboCx DNA polymerase(Stratagene™). Gel-isolated 200-300 by products will be sequenced to alength of 110 bases on the Illumina™ GA II system. The standardIllumina™ image analysis, base calling and processing pipeline will beused to obtain the initial processed sequences. In certain embodiments,only those sequences that pass internal Illumina™ filters(Chastity >0.6) will be stored together with the PHRED-like sequencequality scores in FastQ files. Sequence reads will be trimmed to beforethe first Project Description 12 occurrence of a low quality base (PHREDscore <2). Any remaining cytosine bases in the sequences can beconverted to thymine and the genomic position of this retained in amethyl C coverage file. In certain embodiments, two reference genomescan be generated. In the first reference genome, corresponding to the“Watson” strand, the cytosines can be converted to thymines. In thesecond, corresponding to the Crick strand, guanines can be converted toadenines. The same conversion can be done for the internal controlLambda DNA, which will be analyzed as separate reference genomes for theefficiency of conversion of non-methylated cytosines. The Illuminasequences will be aligned to the two reference genomes with Bowtie(Langmead et al., 2009). In certain embodiments, only sequencing readswith unique starting positions will be scored (a second sequencestarting at the same position will be discarded to minimize unequal PCRamplification distortion of the data). For the Lambda internal control,a conversion rate of nonmethylated cytosines to thymines of greater than99% is expected and will be confirmed in pilot studies and a single laneanalysis of each library (prior to further sequencing of the library),as determined using the internal Lambda DNA control sequences. Theoccurrence of cytosines in the bisulfite-treated Lambda DNA can becomputed as a function of the sequence coverage (each sequence readcounts as coverage of 1). Threshold values will be established to have ap-value of <0.01 for a cytosine occurring by sequencing error orincomplete conversion to uracil.

Two biological replicates can be used for each type of genome analyzed.The coverage can be 10× for each strand. This should be sufficientcoverage to compare the individual biological replicates at mostpositions for individual variation. The combined sequence data from thetwo individuals will be combined for 20× coverage of each strand whencomparing different genotype samples. The individual biologicalreplicates can be used to establish coverage and methylation percentagethresholds to have a False Discovery Rate (FDR) of <0.05 for differencesat specific positions. Selected regions showing methyl C differences canbe analyzed by the traditional bisulfite-PCR-cloning method to validatethe whole genome data and FDR predictions.

Example 7 Quantitative Analysis of Methylation and Phenotypic Variationin Response to MSH1 Suppression

It is possible to exploit the quantitative phenotypic variation thatemerges in an F2 population derived by crossing a MSH1 RNAi-derivedphenotypic variant×wild type. The heritability and quantitativevariation in various sorghum populations subjected to MSH1 suppressionand control sorghum plants described herein can be determined toidentify chromosomal alterations conferring useful traits. In certainembodiments, these methods can entail use of use bisulfite-derived DNASNP polymorphisms identified by sorghum shotgun sequencing experimentsin SNP development and detection. The sorghum genome is about 1628 cM,and we will aim for a SNP marker density of about 1 SNP/10 cM(centimorgans). Therefore, 163 Me-C sites for QTL analysis will beselected on the basis of their differential methylation in the wholegenome analysis of up to five samples types (i.e. (1) wild type, (2)transgenic MSH1 knockdown plants showing dramatically reduced growthrate and delayed flowering, (3) nontransgenic segregants that retain thealtered growth phenotype, (4) F1 plants (as shown in Figure (6) and (5)selected F2 plants exhibiting quantitative variation (FIG. 6)), and foran even 10-cM spacing across the sorghum genome.

DNA from 200 F2 individuals can be bisulfite-treated to create a C/T SNPin the subsequent PCR product. The ratio of C/T will depend on thedegree of Me-C at each methylation site. PCR primers designed to theC-depleted sequences will be used to amplify targeted Me-C SNP regionsin the bisulfite-treated DNA. The C/T polymorphism will be detected on aLightCycler 480 PCR system using Hybprobes™ (Roche, Indianapolis, Ind.,USA). Hybprobes™ use fluorescence resonance energy transfer (FRET)between adjacent probes hybridized to the PCR product and differentialmelting to determine the C/T frequency at the Me-C SNP position.LightCycler™ Probe Design Software (Roche) will be used to design theHybProbes, with the C/T polymorphism in the middle of the sensor probe.The ratio of PCR primers to obtain optimal asymmetric PCR of the Me-Cstrand for hybridization to the HybProbes™ will be experimentallydetermined for each SNP.

Heritability analysis. Up to about two hundred or more F3 families canbe developed in sorghum. DNA can be extracted from each F2 individualgiving rise to each F3 family. A replicated field trial of the F3families can be conducted to perform heritability analysis of theputative epigenetic variation generated by the trans-generationaleffects of the MSH1 RNAi transgene (i.e. MSH1 suppression). For eachspecies, single three meter rows will be arranged in a randomizedcomplete block design with two replications. Populations will be grownin experimental fields.

QTL analysis. Along with the marker data on the 200 F2 individuals, thephenotypic data will be used in a QTL analysis to locate genomic regionsaffected by MSH1 in previous generations that are generating theobserved variation for total biomass and seed yield. A genetic map willbe constructed using segregation data on methylation site changes,followed by standard composite interval mapping.

Example 8 Use of Msh1 Suppression to Alter the Epigenome to ProduceDramatic and Heritable Changes in Plant Growth

Msh1 suppression was used to induce phenotypic and epigenetic variation,and to select derived phenotypes in the crop species Sorghum bicolor(L.) Moench and the model plant Arabidopsis thaliana (L.) Heynh.

FIG. 11 shows the transgene and crossing process that was used in thisstudy for both Arabidopsis and sorghum. In sorghum, all experiments wereconducted with the inbred line Tx430 (F. R. Miller, Crop Sci. 24, 1224,1984), whereas Arabidopsis experiments were carried out in the inbredecotype Columbia-0. MSH1-dr sorghum plants that no longer contain theMSH1-RNAi transgene are restored to normal MSH1 transcript levels;nevertheless, they maintain the altered growth phenotype throughmultiple generations of self-pollination. When crossed reciprocally tothe wildtype inbred Tx430 line, progeny are restored to a normalphenotype. The derived F1 progeny, designated MSH1-epiF1, no longer showthe dwarfed, tillering, late flowering phenotype. In fact, the plantsgrow taller and generally set more seed than the wildtype (FIG. 11A).Self-pollination of the MSH1-epiF1 plants produced an F2 population(MSH1-epiF2) that was strikingly variable in plant phenotype but showedno MSH1-dr phenotype (FIG. 11B-D). A proportion of greenhouse-grownMSH1-epiF3 families did show the MSH1-dr phenotype at a frequency of ca.8% (Table 4), and no dwarf phenotype appeared in the epi-F4 lines.

TABLE 4 Frequency of MSH1-dr phenotype (8.4%) in epi-F3 families derivedfrom sorghum Tx430 MSH1-dr × Tx430 and grown in the greenhouse. Derivedepi-F4 families showed no evidence of the MSH1-dr phenotype (not shown).Mean plant F3 height Tall or family N (cm) wildtype Dwarf 1 10 160 10 02 9 208 9 0 3 10 167 10 0 4 10 189 10 0 5 8 186 7 1 6 10 114 10 0 7 9203 9 0 8 7 102 6 1 9 2 107 2 0 10 9 116 9 0 11 4 89 3 1 12 6 118 6 0 1310 187 10 0 14 8 150 6 2 15 7 81 3 4 16 10 143 7 3 17 5 122 5 0 18 10137 9 1 19 10 98 10 0 19 154 141 13

The F2 plants, and subsequent populations derived by self-pollinating,showed variation for agronomic performance traits, including panicle andplant architecture, tillering time and number, plant height andabove-ground biomass, and yield components of panicle and seed weight(Table 5 for plant height and grain yield). Similarly dramatic changesin growth were observed in Arabidopsis populations derived from crossingthe msh1 mutant with wildtype, followed by selection for the homozygousMSH1/MSH1 F2 plants and serial self-pollination (FIG. 11F-H).

Sorghum MSH1-epiF2, MSH1-epiF3, and MSH1-epiF4 populations grown underfield conditions in 2010 and 2011 permitted larger-scale evaluations ofplant growth changes (Tables 5, 6, 7). Phenotypic distributions weredeveloped from results of two sorghum field experiments, demonstratingpatterns in the MSH1-epiF2 approaching bimodality (FIG. 12). All traitsshowed quantitative patterns of variation. F3 and F4 progenies weretested under both field and greenhouse conditions, displayingheritability for plant height with increasing uniformity among plantseach generation, and response to selection for grain yield, althoughthis trait was subjected to less rigorous selection during growth in thegreenhouse (FIG. 13). These results suggest a high degree ofheritability and selection response for the variation observed.

Altered plant development in sorghum MSH1-dr and Arabidopsis msh1 mutantlines, including variation in growth rate, branching, maturation andflowering, was conditioned by chloroplast changes (see following Example9). We were interested in assessing the relationship of MSH1-epiF2variation to these organellar influences. Arabidopsis MSH1hemi-complementation lines, derived by introducing amitochondrial-versus chloroplast-targeted MSH1 transgene to the msh1mutant line (Y.-Z. Xu et al. Plant Cell 239:3428, 2011), distinguishmitochondrial and chloroplast contributions to the phenomenon. Bothmitochondrial and chloroplast hemi-complementation lines were crossed asfemales to wildtype (Col-0) to produce F1 and F2 progeny. F1 plants fromcrosses to the chloroplast-complemented line produced phenotypes similarto wildtype, although about 25% of the F1 plants showed altered leafcurling and delayed flowering (FIG. 16). This curling phenotype may be aconsequence of MSH1 overexpression, since F1 plants contain both thewildtype MSH1 allele and the transgene. The phenotype resembles effectsof altered salicylic acid pathway regulation, an epigeneticallyregulated process (T. L. Stokes et al. Genes Dev 16, 171, 2002). F1progeny from crosses to the mitochondrial complemented line displayedphenotypic variation in plant growth, with over 30% of the plantsshowing enhanced growth, larger rosette diameter, thicker floral stemsand earlier flowering time, similar to MSH1-epiF3 phenotypes (FIGS. 14A& 17; Table 8). These results were further confirmed in themitochondrial vs. chloroplast-complemented F2 populations (FIG. 14B-E),and suggest that the MSH1-epiF3 enhanced growth changes derive fromrestoring MSH1 function to plants that have undergone the MSH1-drdevelopmental reprogramming phenomenon.

Arabidopsis wildtype and MSH1-epiF3 plants, both Col-0 background, wereinvestigated for evidence of methylome changes that might accompanyheritable MSH1-derived phenotypes. Experiments used sodium bisulfitetreated genomic DNA and genome-wide next-gen sequence analysis (Listeret al. Cell 133, 523, 2008). Methylation changes were extensive, withdifferentially methylated positions involving predominantly CpG sites,with over 91,000 differentially methylated positions in over 1700regions (Table 11, FIG. 15A). The pattern of methylation changes wereconsistent with observed heritability of altered phenotypes, with thelarge proportion of changes in gene coding regions of the genome,resembling data from studies of natural epigenetic variation (C. Beckeret al. Nature 480, 245, 2011; R. J. Schmitz et al. Science 334, 369,2011). Comparison of the non-differential methylation patterns inwildtype and MSH1-epiF3 lines in this study against patterns reported bya recent Arabidopsis study of natural methylation variation (C. Beckeret al. Nature 480, 245, 2011), showed remarkable correspondence ofpattern (FIG. 15B, MSH1-epiF5 line2), confirming consistency of theCol-0 genome methylation analysis between the two studies. Strikingdifferences were evident between the two studies for the regions of thechromosomes enriched for differentially methylated positions; the Beckeret al. analysis of natural variation, shown for illustration purposes inFIG. 15C, showed fairly uniform distribution of differential methylationspanning each chromosome, whereas the MSH1-epiF3 lines revealedirregular patterns of differential methylation that concentrated indiscrete regions of the genome (FIG. 5B, red line). Several DMRs showingchanges in methylation were confirmed by targeted PCR amplification andsequencing of bisulfite-treated DNA intervals (FIG. 18, Table 9). Fromthese results we infer that the developmental variation that accompaniesMSH1 disruption involves pronounced changes in the methylationarchitecture of the plant. The inheritance pattern of the MSH1-drphenotype, showing independence from the transgene and involvement ofnumerous developmental pathways, also indicates that epigenetic changesoccur in the MSH1-dr lines.

TABLE 5 The majority of sorghum F₂ epi-line families consistently show astatistically significant increase in variation (p-value <0.05) in plantheight and grain yield compared to wild-type Tx430. Data were collectedfrom plants grown under field conditions in 2010 and 2011 Plant HeightGrain Yield Per Panicle Mean Std. Error Variance p-value Mean Std. ErrorVariance p-value Year Family (cm) (cm) (cm²) † (g) (g) (g²) † 2010 Tx430132.10 2.42 58.54 — 24.19 0.93 27.11 — 2010 msh1-epi11 F2 165.77 8.402116.67 <0.001 51.29 3.45 368.88 <0.001 2010 msh1-epi15 F2 135.30 5.021182.95 <0.001 33.69 2.47 293.54 <0.001 2010 msh1-epi22 F2 155.96 8.131783.50 <0.001 35.84 2.77 290.84 <0.001 2010 msh1-epi24 F2 140.04 3.401031.38 <0.05 34.35 1.04 185.51 <0.001 2010 msh1-epi28 F2 140.87 3.611130.67 <0.01 23.75 1.58 141.69 <0.001 2011 Tx430 134.50 0.55 64.95 —45.20 0.89 146.49 — 2011 msh1-epi11 F2 186.57 3.93 1912.00 <0.001 53.961.55 272.73 <0.05 2011 msh1-epi15 F2 177.04 2.41 1532.86 <0.001 53.660.94 184.36 <0.05 2011 msh1-epi22 F2 180.73 10.62 1691.50 <0.001 56.622.59 114.08 NS 2011 msh1-epi24 F2 154.78 1.98 1196.96 <0.001 47.92 1.12266.97 <0.001 2011 msh1-epi28 F2 156.91 3.57 1238.75 <0.001 47.49 1.27222.84 <0.05 † p-values based on Levene's test for homogeneity ofvariance in comparison to wild-type Tx430. NS = not significant

TABLE 6 Three of five sorghum epi-F2 line families measured for drybiomass show a statistically significant increase in variation (p-value<0.05) compared to wildtype Tx430. Data were collected from plants grownunder field conditions in 2011. Dry Biomass Yield Mean Std. ErrorVariance Family (g) (g) (g²) p-value† Tx430 53.11 1.94 79.35 —msh1-epi11 F2 85.49 2.77 99.53 NS msh1-epi15 F2 75.08 3.24 252.04 <0.05 msh1-epi22 F2 92.33 7.90 311.83 NS msh1-epi24 F2 68.26 3.54 363.73<0.001 msh1-epi28 F2 66.93 5.79 503.32 <0.001 †p-values based onLevene's test for homogeneity of variance in comparison to wildtypeTx430. NS = not significant

TABLE 7 Sorghum F₄ generation data showing significant differences(p-value <0.05) for many epi-F4 families in plant height (37 of 39lines) and grain yield (11 of 39 lines) compared to wildtype Tx430. Datawere collected from plants grown under field conditions in 2011. GrainYield Per Panicle Plant Height Mean Std. Error Std. Dev. Line Mean (cm)Std. Error (cm) Std. Dev. (cm) p-value * (g) (g) (g) p-value * Tx430134.45 0.56 8.08 — 45.44 0.88 11.95 — 10.3 135.29 1.42 6.515 NS 40.712.17 9.71 NS 12.1 186.24 8.77 47.23 <0.001 44.33 2.73 11.60 NS 12.10238.85 2.01 9.00 <0.001 51.84 2.68 11.99 NS 12.3 220.00 2.55 11.11<0.001 54.86 3.15 13.72 NS 14.1 187.20 5.72 25.59 <0.001 56.59 3.5014.87 <0.05 15.2 222.75 1.76 8.63 <0.001 33.88 1.74 8.52 <0.001 17.2174.52 6.55 36.49 <0.001 61.25 2.54 11.05 <0.001 17.3 192.54 5.66 27.72<0.001 47.02 1.74 8.16 NS 2a-9 216.00 4.44 19.34 <0.001 48.12 3.40 14.41NS 2b-1 217.83 3.41 14.49 <0.001 43.88 4.29 17.69 NS 2b-3 221.24 2.108.67 <0.001 54.82 3.94 16.25 NS 2b-4 217.44 2.65 10.60 <0.001 44.75 3.3612.08 NS 2b-5 231.32 3.46 15.07 <0.001 53.40 3.00 12.70 NS 2b-6 229.901.49 6.67 <0.001 50.52 2.43 10.87 NS 2b-8 231.21 1.61 7.89 <0.001 39.952.71 13.27 NS 2b-10 207.80 4.01 17.94 <0.001 66.94 3.99 17.84 <0.0013a-1 226.79 2.74 11.93 <0.001 44.39 3.09 12.73 NS 3a-2 141.10 1.78 7.97<0.05 46.61 2.61 11.96 NS 3a-6 233.14 1.63 7.48 <0.001 44.35 2.24 10.27NS 3a-7 190.29 9.58 43.89 <0.001 40.30 3.91 15.15 NS 3b-1 219.44 2.5110.68 <0.001 41.47 3.69 13.82 NS 3b-2 216.65 2.49 11.12 <0.001 52.141.96 8.77 <0.05 3b-3 210.28 3.34 14.17 <0.001 39.99 3.69 11.08 NS 3b-4207.64 4.72927 22.18 <0.001 51.17 2.27 10.39 NS 3b-7 223.41 2.3531259.70 <0.001 53.10 3.45 14.22 NS 3b-10 234.14 2.170879 8.12 <0.001 43.043.22 9.10 NS 4a-1 213.07 3.164821 11.84 <0.001 60.54 6.29 22.66 <0.014a-2 217.67 7.862307 30.45 <0.001 52.33 3.40 10.77 NS 4a-4 225.565.02882 21.34 <0.001 52.11 3.58 14.78 NS 4a-7 233.28 2.471809 10.49<0.001 41.28 2.15 8.87 NS 4a-8 200.31 7.515885 38.32 <0.001 48.04 2.6011.05 NS 4b-10 133.06 1.403771 5.62 NS 63.96 3.39 13.55 <0.001 5a-1216.48 4.470243 17.88 <0.001 68.90 4.72 18.28 <0.001 5a-2 219.052.415699 11.07 <0.001 43.20 1.64 7.49 NS 5a-3 220.58 2.359566 8.17<0.001 58.30 2.66 9.58 <0.001 5a-5 214.67 3.178769 13.49 <0.001 52.162.60 11.02 NS 5a-6 216.94 3.335935 13.75 <0.001 53.35 2.80 11.21 NS 5a-8212.90 3.568814 19.55 <0.001 52.74 1.41 7.74 <0.001 5a-9 227.29 2.31880810.63 <0.001 59.80 3.36 15.38 <0.01 * p-values based on max-t test formultiple comparison of means (Dunnett contrasts) using heteroscedasticconsistent covariance estimation (E. Herberich et al. PLoS One. 5(3):e9788 (2010)), against wildtype Tx430. NS = not significant

TABLE 8 Analysis of phenotype data from individual Arabidopsis F₂families derived by crossing hemi-complementation lines × Col-0wildtype. SSU-MSH1 refers to lines transformed with the plastid-targetedform of MSH1; AOX-MSH1 refers. to lines containing the mitochondrial-targeted form of the MSH1 transgene. In all genetic experiments usinghemi-complementation, presence of the transgene was confirmed with aPCR-based assay. Rosette diameter Fresh biomass Population Mean (cm) NStd Error Std Dev p-value Mean (g) N Std Error Std Dev p-value AOX-MSH111.07 36 0.37 2.23 <0.001 8.86 10 0.47 1.33 NS SSU-MSH1 11.76 18 0.261.10 <0.001 10 10 0.55 1.55 NS Col-0 12.98 42 0.24 1.59 — 9.45 10 0.431.36 — F-2 12.83 21 0.34 1.57 NS 15.07 10 0.66 2.07 <0.001 (AOX-MSH1 ×Col-0)F-22 13.82 21 0.42 1.92 <0.10 14.62 10 0.92 2.24 <0.001 (AOX-MSH1× Col-0)F-28 14.85 21 0.31 1.42 <0.001 13.27 10 0.70 1.99 <0.001(AOX-MSH1 × Col-0)F-26 12.82 20 0.25 1.12 NS 10.57 10 0.66 1.74 NS(SSU-MSH1 × Col-0)F-29 11.9 21 0.27 1.25 <0.001 10.5 10 0.45 1.19 NS Pvalues are based on two-tailed Student t-test comparing to Col-0 NS =Not Significant

TABLE 9 Sample differential methylation data for four DMRs, derived byPCR- based analysis of bisulfite-treated DNA from Arabidopsis wildtypeCol-0 and MSH1-epiF3 lines. Region No. % % size DMP in methylationmethylation AGI Gene (bp) region Site in Col-0 in F3 AT5G67120RING/U-box 200 8 1 20% 86% superfamily protein 2 30% 86% 3 20% 100% 430% 100% 5 30% 100% 6 30% 100% 7 30% 86% 8 20% 100% AT1G20690 SWI-SNF100 6 1 27% 75% related protein 2 27% 83% 3 18% 100% 4 18% 92% 5 18% 83%6 63% 92% AT3G27150 Target of MIR2111-5p region 1 200 9 1 0 58% 2 0 67%3 0 92% 4 0 100% 5 0 83% 6 0 92% 7 0 67% 8 0 92% 9 0 75% region 2 250 171 0 100% AT5G67120 RING/U-box 200 8 1 20% 86% superfamily protein 2 0100% 3 58% 100% 4 0 100% 5 0 100% 6 0 100% 7 0 100% 8 0 73% 9 8% 100% 108% 82% 11 0 82% 12 8% 100% 13 0 91% 14 0 82% 15 0 82% 16 0 73% 17 0 91%

TABLE 10 Primers used in the study Primer name Sequence (SEQ ID NO:)For bisulfite sequencing: AT5G67120RING-F 5'-TTTTTAGGAATTATTGAGTATTATTGA-3' (SEQ ID NO: 17) AT5G67120RING-R 5'-AAATAAAAATCATACCCACATCC C-3'(SEQ ID NO: 18) AT1G20690SWI-F 5'-TGTTGAATTATTAAGATATTTAA GAT-3'(SEQ ID NO: 19) AT1G20690SWI-R 5'-TCAACCAATAAAAATTACCATCT AC-3'(SEQ ID NO: 20) AT3g271501stMir2-F 5'-TAAGTTTTTTTTAAGAGTTTGTA TTTGTAT-3'(SEQ ID NO: 21) AT3g271501stMir2-R 5'-TAAAAATAATCAAAACCTAACTT AC-3'(SEQ ID NO: 22) AT3g271502ndMir2-F 5'-ATTGTTTATTAAATGTTTTTTAG TT-3'(SEQ ID NO: 23) AT3g271502ndMir2-R 5'-CTAACAATTCCCAAAACCCTTAT C-3'(SEQ ID NO: 24) For PCR assay of MSHI-RNAi transgene: RNAi-F5'-GTGTACTCATCTGGATCTGTATT G-3' (SEQ ID NO: 25) RNAi-R5'-GGTTGAGGAGCCTGAATCTCTGA AC-3' (SEQ ID NO: 26)

TABLE 11 Genome-wide 5-methylcytosine analysis in Arabidopsis Col-0 andMSH1-epiF3 plants. Background CpG CHG CHH Mapped 4,382,312 4,749,45119,727,351 Methylated 950,806 589,084 1,062,553 DMPs 91,150 10,324 1,789DMRs 1,770 93 15

Plant phenotypes derived from crossing the MSH1-dr selections towildtype did not appear to resemble those reported from other types ofinduced methylation changes, even though methylome changes were evidentin the resulting populations. EpiRIL populations produced from crossesinvolving the Arabidopsis met1 mutant give rise to a variety of variantphenotypes (J. Reinders et al., Genes Dev. 23, 939 (2009). These earlierstudies do not, however, report the enhanced vigor, markedly largerplant and stem size, or greater seed production that is seen with MSH1manipulation.

The materials and methods used in this Example are as described below.

Plant Materials and Growth Conditions

Arabidopsis Col-0 and msh1 mutant lines were obtained from theArabidopsis stock center and grown in metro mix with 12 hr daylight at22° C. MSH1-epi lines were derived by crossing MSH1-dr lines with wildtype plants. Arabidopsis plant biomass and rosette diameter weremeasured for 4-week-old plants. Arabidopsis flowering time was measuredas date of first visible flower bud appearance. For hemi-complementationcrosses, mitochondrial (AOX-MSH1) and plastid (SSU-MSH1) complementedhomozygous lines were crossed to Columbia-0 wildtype plants. Each F1plant was genotyped for transgene and the wildtype MSH1 allele andharvested separately. Three F2 families from AOX-MSH1×Col-0 and two F2families from SSU-MSH1×Col-0 were evaluated for growth parameters. Allfamilies were grown under the same conditions, and biomass, rosettediameter and flowering time were measured. Two-tailed Student t-test wasused to calculate p-values.

The sorghum germplasm used in these experiments was derived from Tx430,an inbred sorghum line (Miller, 1984). Several T3 sorghum siblings werederived from a single MSH1-dr plant, grown under greenhouse conditionsand designated GAII1-GAII30. Each of the lines were confirmed to betransgene nulls. Six of them, GAII11, GAII15, GAII22, GAII24, GAII25,and GAII28 were used as females in crosses to wild type inbred Tx430 toderive F1 seed. Three additional plants, GAII22, GAII23, and GAII27 wereused as males in reciprocal crosses. Day temperature in the greenhousewas 79 to 83° F., and night was 69 to 73° F. Plants were grown undershort (10-hr) daylength.

F1 progenies were grown under the same greenhouse conditions, withprogenies ranging in size from 5-19 individuals. Derived T4 progenieswere grown from the six maternal msh1-dr plants used to derive F1s(GAII11, GAII15, GAII22, GAII24, GAII25, and GAII28), with populationsranging in size from 15-19 individuals. Self-pollinated seed of every F1plant was harvested individually to derive the corresponding F2families.

Field Experiments

During the summers of 2010 and 2011, F2 families were grown in two fieldexperiments established under rainfed conditions at the HavelockExperiment Station of University of Nebraska in Lincoln. Experimentswere arranged in an incomplete block design, with the 2010 experimentconsisting of one replication with 15 blocks and 30 entries per block(30×15 alpha lattice). Individual lines were planted in a singlepanicle-per-row plan, with a single row plot of 5-m length and 0.75-mbetween-row spacing. The F3 seed was harvested from individual plants.

The 2011 experiment comprised seven blocks of 28 entries each (28×7alpha lattice), with two replications fertilized with supplementalnitrogen at a dosage of 100 kg/ha. Forty eight samples from the 2010experiment were selected to comprise the F3. These samples were derivedfrom all six original crosses and included high and low F2 grain yieldvalues. In addition, a greenhouse-grown subgroup of 17 F3 samples wereselected, based on dry panicle weight, to derive F4 seed. Thus, the 2011field experiment comprised 48, 77, and 42 entries corresponding to theF2, F3 and F4 generations, respectively, with wildtype Tx430 as control.

Sorghum Phenotypic Assessment

In 2010 and 2011 field experiments, the sorghum phenotypic traitsrecorded included plant height (PH), in cm from ground to panicle tip,panicle length (PL), in cm from panicle base to tip, fresh and drypanicle weight (FPW and DPW) (g), fresh and dry biomass yield (FBY andDBY) (g), and net grain yield (NGY) (g). Sample size for PH, PL, FPW,DPW and NGY varied from five to ten random, inner-row plants per row.Healthy, well-shaped heads were bagged before anthesis for selfing, andharvested after physiological maturity, when FPW was measured. Thesamples were dried at 80° F. for 30 days prior to measuring DPW and NGY.Biomass samples consisted of a three-plant sample, bagged and weighedafter cutting to obtain FBW. Plants were random, inner-row selections,and samples were completely dried at 160° F. over 15 days for DBW.

PCR Assay for RNAi Transgene.

PCR assay for MSH1-RNAi transgene presence in sorghum materials usedprimers listed in Table S7. The reaction conditions were: 95° C. 5 min,30 cycles of 95° C. 30 s, 55° C. 1 min, 72° C. 2 min; final extensionwas at 72° C. 10 min. Positive and negative controls were included froma confirmed transgenic line and wildtype Tx430, respectively.

Bisulfite Treated Genomic Library Construction and Sequencing

Arabidopsis genomic DNA (ca 15 ug) prepared from Col-0 and epi-F3 plantswas sonicated to a peak range of 200 bp to 600 bp, phenol/chloroformpurified and ethanol precipitated. Sonicated DNA (ca 12 ug) was treatedwith Mung Bean Nuclease (New England Biolabs), phenol/chloroformextracted and ethanol precipitated. Mung Bean Nuclease-treated genomicDNA (ca 3 ug) was end-repaired and 3′ end-adenylated with IlluminaGenomic DNA Samples Prep Kit (Illumina, San Diego Calif.). Theadenylated DNA fragment was then ligated to methylation adapters(Illumina, San Diego, Calif.). Samples were then column purified andfractionated in agarose. A fraction of 280 bp to 400 bp was gel purifiedwith the QIAquick Gel Purification kit (Qiagen, Valencia, Calif.).Another 3 ug of Mung Bean Nuclease treated genomic DNA was used torepeat the process, and the two fractions were pooled and subjected tosodium bisulfite treatment with the MethylEasy Xceed kit (Human GeneticSignatures Pty Ltd, North Ryde, Australia) according to manufacturer'sinstructions. Three independent library PCR enrichments were carried outwith 10 ul from a total of 30 ul bisulfate treated DNA as inputtemplate. The PCR reaction mixture was 10 ul DNA, 5 ul of 10× pfuTurboCx buffer, 0.7 ul of PE1.0 primer, 0.7 ul PE2.0 primer, 0.5 ul of dNTP(25 mM), 1 ul of PfuTurbo Cx Hotstart DNA Polymerase (Stratagene, SantaClara, Calif.), and water to a total volume of 50 ul. The PCR parameterswere 950 C for 2 min, followed by 12 cycles of 950 C 30 sec, 650 C 30sec and 720 C 1 min, then 720 C for 5 min. PCR product wascolumn-purified and an equal volume from each PCR reaction was pooledtogether to a final concentration of 10 nM.

Libraries were DNA sequenced on the Illumina Genome Analyzer II withthree 36-cycle TrueSeq sequencing kits v5 to read 116 nucleotides ofsequence from a single end of each insert (V8 protocol).

Bisulfite Treatment of DNA for PCR Analysis

Arabidopsis genomic DNA was bisulfite treated using the MethylEasy Xceedkit according to manufacturer's instructions. PCR was performed usingprimers listed in Table S7, and the PCR products were cloned (Topo TAcloning kit, Invitrogen) and DNA-sequenced. Sequence alignment wasperformed using the T-Coffee multiple sequence alignment server (CNotredame, et al., J Mol Biol. 302:205-217, 2000).

DNA Sequence analysis and identification of differentially methylatedcytosines (DMCs).

Fastq files were aligned to the TAIR10 reference genome using Bismark (FKrueger, S R Andrews. Bioinformatics 27:1571-1572 (2011), which was alsoused to determine the methylation state of cytosines. One mismatch wasallowed in the first 50 nucleotides of the read. Bismark only retainsreads that can be uniquely mapped to a location in the genome.

Only cytosine positions identified as methylated in at least two readsfor at least one of the genotypes and sequenced at least four times ineach of the genotypes were used for the identification of DMCs. Forthese cytosine positions, the number of reads indicating methylation ornon-methylation for each genotype was tabulated using R(http://www.r-project.org). Fisher's exact test was carried out fortesting differential methylation at each position. Adjustment formultiple testing over the entire genome was done as suggested in Storeyand Tibshirani (J D Storey, R Tibshirani. Proc. Natl. Acad. Sci. USA100:9440-9445 (2003) and a false discovery rate (FDR) of 0.05 was usedfor identifying differentially methylated cytosines. Methylome sequencedata have been uploaded to the Gene Expression Omnibus with accessionnumber GSE36783.

Mapping DMCs to genomic context and identifying differentiallymethylated regions (DMRs).

TAIR10 annotation (available on the internet ftp site“ftp.arabidopsis.org/home/tair/Genes/TAIR10_genome_release/TAIR10_gff3”)was used to determine the counts for DMCs or nondifferentiallymethylated cytosines in gene coding regions, 5′-UTRs, 3′-UTRs, introns,pseudogenes, non-coding RNAs, transposable element genes, and intergenicregions. Intergenic regions were defined as regions not corresponding toany annotated feature.

For each methylation context (CpG, CHG, CHH), the genome was scanned forregions enriched in DMCs using a 1-kb window in 100-bp increments.Windows with at least four DMCs were retained and overlapping windowswere merged into regions. Regions with at least 10 DMCs were retainedwith the boundary trimmed to the furthest DMCs in the region. Fisher'sexact test was then performed for each region by merging allmethylated/non-methylated read counts at all cytosine positions in theregion. Adjusting for all tested regions, the FDR is controlled at 0.1.

Example 10 Summary Table of Nucleic Acid Sequences and SEQ ID NO

TABLE 12 Nucleotide Sequences provided in the Sequence Listing SEQInternet Accession ID Information NO Comments The ArabidopsisInformation Resource 1 Arabidopsis (TAIR) MSH1 1009043787 Full lengthcDNA (DNA on the internet (world wide web) at sequence) arabidopsis. orgThe Arabidopsis Information Resource 2 Arabidopsis (TAIR) MSH1 Protein(amino acid 1009118392 sequence) on the internet (world wide web) atarabidopsis. org NCBI AY856369 3 Soybean MSH1 on the world wide webat >gi|61696668|gb|AY856369.1| ncbi.nlm.nih.gov/nuccore Glycine max DNAmismatch repair protein (MSH1) complete cds; (DNA sequence) NCBIAccession 4 Zea mays MSH1 AY856370 gi|61696670|gb|AY856370.1| on theworld wide web at Zea mays DNA mismatch repair ncbi.nlm.nih.gov/nuccoreprotein (MSH1), complete cds; (DNA sequence) NCBI Accession 5 TomatoMSH1 AY866434.1 >gi|61696672|gb|AY866434.1| on the world wide web atLycopersicon esculentum DNA ncbi.nlm.nih.gov/nuccore mismatch repairprotein (MSH1), partial cds; (DNA sequence) NCBI 6 Sorghum MSH1XM002448093.1 >gi|242076403:1-3180 Sorghum on the world wide web atbicolor hypothetical protein; ncbi.nlm.nih.gov/nuccore (DNA sequence)Os04g42784.1 7 Rice (Oryza sativa) MSH1 Rice Genome Annotation Project -MSU coding sequence (DNA Rice Genome Annotation (Osa1) Release sequence)6.1 Internet address rice.plantbiology.msu.edu/index.shtml Brachypodium8 Brachypodium Bradi5g15120.1 MSH1 coding region (DNA On the world wideweb at sequence) gramene.org/Brachypodium_distachyon/Gene/Summary?db=core;g=BRADI5G15120;r =5:18500245- 18518223;t=BRADI5G15120.1GSVIVT01027931001 9 Vitis Vinifera On the world wide web at MSH1 cDNA(DNA sequence) genoscope.cns.fr/spip/Vitis-vinifera-e.htmlCucsa.255860.1 10 Cucumber (Cucumis sativa) On the internet (world wideweb) at MSH1 coding sequence; (DNA phytozome.net/ sequence) TOM-CD1F 11Primer (DNA sequence) TOM-CD1R 12 Primer (DNA sequence) At4g02840 13second intron of the Arabidopsis The Arabidopsis Information Resourcesmall nuclear riboprotein (TAIR) (At4g02840); (DNA sequence) on theinternet (world wide web) at arabidopsis. org GenBank Accession 14Cotton (Gossypium hirsutum) ES831813.1 MSH1 partial cDNA sequence on theworld wide web at (EST); (DNA sequence) ncbi.nlm.nih.gov/nucest Primer15 Primer (DNA sequence) zm-msf8 Primer 16 primer(DNA sequence) zm-msr8AT5G67120RING-F 17 primer(DNA sequence) AT5G67120RING-R 18 primer(DNAsequence) AT1G20690SWI-F 19 primer(DNA sequence) AT1G20690SWI-R 20primer(DNA sequence) AT3g271501stMir2-F 21 primer(DNA sequence)AT3g271501stMir2-R 22 primer(DNA sequence) AT3g271502ndMir2-F 23primer(DNA sequence) AT3g271502ndMir2-R 24 primer(DNA sequence) RNAi-F25 primer(DNA sequence) RNAi-R 26 primer(DNA sequence) AT3G27150 27 DNAsequence The Arabidopsis Information Resource (TAIR) on the internet(world wide web) at arabidopsis.org Col0-MIR2-2 28 DNA sequence(bisulfite sequencing) Col0-MIR2-3 29 DNA sequence (bisulfitesequencing) Col0-MIR2-4 30 DNA sequence (bisulfite sequencing)Col0-MIR2-5 31 DNA sequence (bisulfite sequencing) Col0-MIR2-6 32 DNAsequence (bisulfite sequencing) Col0-MIR2-10 33 DNA sequence (bisulfitesequencing) Col0-MIR2-11 34 DNA sequence (bisulfite sequencing)Col0-MIR2-12 35 DNA sequence (bisulfite sequencing) Col0-MIR2-26 36 DNAsequence (bisulfite sequencing) Col0-MIR2-27 37 DNA sequence (bisulfitesequencing) Col0-MIR2-28 38 DNA sequence (bisulfite sequencing)Col0-MIR2-29 39 DNA sequence (bisulfite sequencing) F3-Mir2-1 40 DNAsequence (bisulfite sequencing) F3-Mir2-2 41 DNA sequence (bisulfitesequencing) F3-Mir2-4 42 DNA sequence (bisulfite sequencing) F3-Mir2-543 DNA sequence (bisulfite sequencing) F3-Mir2-7 44 DNA sequence(bisulfite sequencing) F3-Mir2-11 45 DNA sequence (bisulfite sequencing)F3-Mir2-12 46 DNA sequence (bisulfite sequencing) F3-Mir2-15 47 DNAsequence (bisulfite sequencing) F3-Mir2-16 48 DNA sequence (bisulfitesequencing) F3-Mir2-27 49 DNA sequence (bisulfite sequencing) F3-Mir2-2850 DNA sequence (bisulfite sequencing) Brassica Locus Bra015033 (Msh1ortholog) 51 DNA sequence of the Brassica Available on the internet(world wide web) rapa Msh1 ortholog atchibba.agtec.uga.edu/duplication/index/details ?lc=Bra015033 Wheat LocusQ8RVT1 52 WHEAT MutS homolog 7 GenBank Accession No.: AF354709.1(Fragment) Partial coding sequence Available on the internet (world wideweb) at ncbi.nlm.nih.gov/nuccore/AF354709

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Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles.

Although the materials and methods of this invention have been describedin terms of various embodiments and illustrative examples, it will beapparent to those of skill in the art that variations can be applied tothe materials and methods described herein without departing from theconcept, spirit and scope of the invention. All such similar substitutesand modifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1. A method for producing a plant exhibiting a useful trait comprisingthe steps of: a. suppressing expression of MSH1 gene(s) in a firstparental plant or plant cell; b. outcrossing the parental plant of step(a), progeny of said parental plant of step (a), a plant obtained fromsaid plant cell of step (a), or progeny of a plant obtained from saidplant cell of step (a) to a second plant wherein MSH1 had not beensuppressed; c. screening a population of progeny plants obtained fromsaid outcross of step (b) for at least one useful trait, wherein aportion of said population of progeny plants express MSH1; and, d.selecting a progeny plant comprising said trait that expresses MSH1,wherein said trait is heritable and reversible.
 2. The method of claim1, wherein said trait is associated with one or more altered chromosomalloci.
 3. The method of claim 1, wherein said trait is associated withone or more mutated chromosomal loci.
 4. The method of claim 1, whereinsaid method further comprises the step of producing seed from: i) aselfed progeny plant of step (d), ii) an out-crossed progeny plant ofstep (d), or, iii) from both of a selfed and an out-crossed progenyplant of step (d).
 5. The method of claim 4, wherein said method furthercomprises the step of assaying said seed or plants grown from said seedfor the presence of said trait.
 6. The method of claim 1, wherein saidfirst parental plant or plant cell comprises a transgene that cansuppress expression of MSH1.
 7. (canceled)
 8. The method of claim 1,wherein said first parental plant or plant cell is obtained by crossingan female plant with a distinct male plant, wherein at least one of saidfemale or male plants comprise a transgene that suppresses expression ofthe endogenous MSH1 gene of said parental plant(s), and wherein saidplants were isogenic inbred lines prior to introduction of saidtransgene.
 9. The method of claim 1, wherein said first parental plantor plant cell was isogenic to said second parental plant prior tosuppression of MSH1 in said first parental plant or plant cell.
 10. Themethod of claim 1, wherein said trait is selected from the groupconsisting of yield, male sterility, non-flowering, biotic stress, andabiotic stress.
 11. The method of claim 10, wherein said trait is notcaused by substoichiometric shifting (SSS) in mitochondria of saidprogeny plant.
 12. (canceled)
 13. The method of claim 1, wherein saidprogeny plant in step (d) or progeny thereof exhibit an improvement insaid trait in comparison to a plant that had not been subjected tosuppression of MSH1 expression but was otherwise isogenic to said firstparental plant or plant cell.
 14. (canceled)
 15. The method of claim 14,wherein said plant is a crop plant is selected from the group consistingof corn, soybean, cotton, canola, wheat, rice, tomato, tobacco, millet,and sorghum.
 16. (canceled)
 17. The method of claim 15, wherein saidcrop plant is sorghum and said trait is selected from the groupconsisting of panicle length, panicle weight, dry biomass, andcombinations thereof.
 18. A plant produced by the methods of claim 1,wherein said plant exhibits an improvement in at least one useful traitin comparison to a plant that had not been subjected to suppression ofMSH1 expression but was otherwise isogenic to said first parental plantor plant cell.
 19. (canceled)
 20. Seed obtained from the plant of claim18, wherein the seed or a plant obtained therefrom exhibits theimprovement in at least one useful trait.
 21. A processed product fromthe plant of claim 18 or from the seed therefrom, wherein said productcomprises a detectable amount of a chromosomal DNA, a mitochondrial DNA,a plastid DNA, or any combination thereof.
 22. (canceled)
 23. A methodfor producing a lot of seed, comprising the steps of selfing apopulation of plants of claim 18, and harvesting seed therefrom, whereinthe harvested seed or a plant obtained therefrom exhibits theimprovement in at least one useful trait.
 24. A method for identifyingone or more altered chromosomal loci in a plant that can confer a usefultrait comprising the steps of: a) comparing one or more chromosomalregions in a reference plant that does not exhibit said useful trait toone or more corresponding chromosomal regions in a test plant that doesexhibit said useful trait, wherein said test plant expresses MSH1 andwas obtained from a parental plant or plant cell wherein MSH1 had beensuppressed; and, b) selecting for one or more altered chromosomal locipresent in said test plant that are absent in said reference plant andthat are associated with said useful trait.
 25. The method of claim 24,wherein said altered chromosomal loci comprise a chromosomal DNAmethylation state, a post-translation modification of a histone proteinassociated with a chromosomal locus, or any combination thereof.
 26. Themethod of claim 24, wherein said selection comprises isolating a plantor progeny plant comprising said altered chromosomal locus or obtaininga nucleic acid associated with said altered chromosomal locus.
 27. Themethod of claim 24, wherein said reference plant and said test plant areboth obtained from a population of progeny plants obtained from aparental plant or plant cell wherein MSH1 had been suppressed.
 28. Themethod of claim 24, wherein both said reference plant and said parentalplant or plant cell were isogenic prior to suppression of MSH1 in saidparental plant or plant cell.
 29. The method of claim 24, wherein saiduseful trait is selected from the group consisting of yield, malesterility, non-flowering, biotic stress resistance, abiotic stressresistance, enhanced lodging resistance, enhanced growth rate, enhancedbiomass, enhanced tillering, enhanced branching, delayed flowering time,and delayed senescence.
 30. An altered chromosomal locus of a plantidentified by the method of claim
 24. 31. A plant comprising the alteredchromosomal locus of claim
 30. 32. A method for producing a plantexhibiting a useful trait comprising the steps of: a. introducing achromosomal modification associated with a useful trait into a plant,wherein said chromosomal modification comprises a altered chromosomallocus induced by MSH1 suppression associated with said useful trait, atransgene that provides for the same genetic effect as an alteredchromosomal locus induced by MSH1 suppression associated with saiduseful trait, or a chromosomal mutation that provides for the samegenetic effect as an altered chromosomal locus induced by MSH1suppression associated with said useful trait; and, b. selecting for aplant that comprises said chromosomal modification and exhibits saiduseful trait.
 33. The method of claim 32, wherein said method furthercomprises the step of producing seed from: i) a selfed progeny plant ofsaid selected plant of step (b), ii) an out-crossed progeny plant ofsaid selected plant of step (b), or, iii) from both of a selfed and anout-crossed progeny plant of said selected plant of step (b).
 34. Themethod of claim 32, wherein said chromosomal modification comprises analtered chromosomal locus and wherein said plant is selected by assayingfor the presence of a chromosomal DNA methylation state, apost-translation modification of a histone protein associated with achromosomal locus, or any combination thereof, that is associated withsaid altered chromosomal locus.
 35. The method of claim 32, wherein saidchromosomal modification comprises said transgene or said chromosomalmutation and wherein said plant is selected by assaying for the presenceof said transgene or said chromosomal mutation.
 36. The method of claim32, wherein said plant is selected by assaying for the presence of saiduseful trait.
 37. The method of claim 32, wherein said chromosomalmodification comprises an altered chromosomal locus and wherein saidaltered chromosomal locus comprises a chromosomal DNA methylation state,a post-translation modification of a histone protein associated with achromosomal locus, or any combination thereof.
 38. The method of claim32, wherein said altered chromosomal locus has a genetic effect thatcomprises a reduction in expression of a gene and wherein saidchromosomal modification comprises a transgene or a chromosomal mutationthat provides for a reduction in expression of said gene.
 39. (canceled)40. The method of claim 32, wherein said altered chromosomal locus has agenetic effect that comprises an increase in expression of a gene andwherein said chromosomal modification comprises a transgene or achromosomal mutation that provides for an increase in expression of saidgene.
 41. The method of claim 32, wherein said useful trait is selectedfrom the group consisting of yield, male sterility, non-flowering,biotic stress resistance, and abiotic stress resistance.
 42. The methodof claim 32, wherein said useful trait is selected from the groupconsisting of enhanced lodging resistance, enhanced growth rate,enhanced biomass, enhanced tillering, enhanced branching, delayedflowering time, and delayed senescence.
 43. A plant made by the methodof claim 32.